Anti-CTLA4 antibodies and methods of making and using the same

ABSTRACT

Provided herein are cross-reactive antibodies (or antigen binding fragments thereof) that bind to human CTLA4, activatable antibodies that bind to human CTLA4, nucleic acid molecules encoding the same, pharmaceutical compositions thereof, and methods of their therapeutic use (e.g., for treatment of cancer).

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of InternationalApplication No. PCT/CN2018/075064, filed on Feb. 2, 2018, which isincorporated herein by reference in its entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 695402000520SEQLIST.TXT,date recorded: Feb. 1, 2019, size: 102 KB).

FIELD OF THE INVENTION

The present disclosure relates to cross-reactive antibodies that bind tohuman Cytotoxic T-lymphocyte Protein 4 (CTLA4),precision/context-dependent activatable antibodies that bind to humanCTLA4, nucleic acids encoding the same, pharmaceutical compositionsthereof, and their therapeutic use.

BACKGROUND

CTLA4 is a member of the immunoglobulin (Ig) superfamily of proteinsthat acts to downregulate T-cell activation and maintain immunogenichomeostasis. It has been shown that in vivo antibody-mediated blockadeof CTLA4 enhanced anti-cancer immune responses in a syngeneic murineprostate cancer model (Kwon et al. (1997) Proc Natl Acad Sci USA,94(15):8099-103). In addition, blockade of CTLA4 function was shown toenhance anti-tumor T cell responses at various stages of tumor growth intumor-bearing mice (Yang et al. (1997) Cancer Res 57(18):4036-41;Hurwitz et al. (1998) Proc Natl Acad Sci USA 95 (17):10067-7). However,the development of antibody-based therapeutics suitable for human useremains difficult, as translation from pre-clinical animal models tohuman safety is often poor. Accordingly, a need exists for anti-CTLA4antibodies that are cross-reactive among different species, such ashumans and experimental animals (e.g., mouse, monkey, rat, etc.), toconcurrently enable animal model studies and provide suitable humantherapeutic candidates. In addition, a need exists for the developmentof safer anti-CTLA4 antibodies that are only active in certain contexts,such as in the protease-rich tumor microenvironment.

All references cited herein, including patent applications, patentpublications, non-patent literature, and UniProtKB/Swiss-Prot/GenBankAccession numbers are herein incorporated by reference in theirentirety, as if each individual reference were specifically andindividually indicated to be incorporated by reference.

BRIEF SUMMARY

To meet the above and other needs, disclosed herein are antibodies(e.g., cross-reactive antibodies), and antigen binding fragmentsthereof, that bind to human CTLA4. The anti-CTLA4 antibodies, or antigenbinding fragments thereof, of the present disclosure possessed at leastone (e.g., one, some, or all) of the following functional properties:(a) bind to human, cynomolgus monkey, mouse, rat, and/or dog CTLA4 witha K_(D) of 500 nM or less; (b) have antagonist activity on human CTLA4;(c) do not bind to human PD-1, PD-L1, PD-L2, LAG3, TIM3, B7-H3, CD95,CD120a, OX40, CD40, BTLA, VISTA, ICOS, and/or B7-H4 at concentration upto 100 nM; (d) are cross-reactive with monkey, mouse, rat, and/or dogCTLA4; (e) induces ADCC effects (e.g., on Tregs); (f) activates humanPBMCs (e.g., stimulates secretion of IL-2 and/or IFNγ); (g) are capableof inhibiting tumor cell growth and establishing immune memory againsttumor cells; (h) have therapeutic effect on a cancer; and (i) blockbinding of human CTLA4 to human CD80 and/or human CD86 (see Examples 1-5below).

Disclosed herein are precision/context-dependent activatable antibodiesthat bind to human CTLA4 when in active form but not in inactive form,i.e., the activatable antibodies bound to CTLA4 (were active) only aftercleavage of the cleavable moiety (CM) to remove the masking moiety (MM).In some embodiments, the discovered masking moieties (MMs) describedherein were capable of efficiently masking antibody activity and/orreducing or completely inhibiting antigen binding, while in someembodiments being devoid of the chemically labile residues methionineand/or tryptophan. Furthermore, the activatable antibodies identifiedand described herein are as efficient at treating multiple cancer typesas their parental antibody, while having significantly reducedcytotoxicity in susceptible animals (NOD mice).

Accordingly, in one aspect, provided herein is an anti-CTLA4 antibody(e.g., human antibodies) that binds human CTLA4 and is cross-reactivewith a CTLA4 polypeptide from at least one non-human animal selectedfrom the group consisting of cynomolgus monkey, mouse, rat, and dog. Insome embodiments, the antibody binds to cynomolgus monkey CTLA4 andmouse CTLA4. In some embodiments that may be combined with any of thepreceding embodiments, the antibody binds to human CTLA4, cynomolgusmonkey CTLA4, mouse CTLA4, rat CTLA4, and/or dog CTLA4 with adissociation constant (K_(D)) of about 350 nM or less (e.g., about 300nM or less, about 200 nM or less, about 100 nM or less, about 50 nM orless, or about 10 nM or less). In some embodiments, the K_(D) ismeasured by surface plasmon resonance (SPR). In some embodiments,binding of the antibody to CTLA4 induces antibody-dependent cellcytotoxicity (ADCC) against a CTLA4-expressing cell. In someembodiments, binding of the antibody to CTLA4 induces ADCC against aTreg cell. In some embodiments, binding of the anti-CTLA4 antibodydescribed herein induces antibody-dependent cell cytotoxicity (ADCC)against a CTLA4-expressing human cell or a human Treg cell, wherein theADCC activity of the anti-CTL4 antibody is higher than the ADCC activityof ipilimumab in vitro, and wherein both antibodies comprise wild typehuman IgG1 Fc region. In some embodiments, binding of the anti-CTLA4antibody described herein induces antibody-dependent cell cytotoxicity(ADCC) against a CTLA4-expressing human cell or a human Treg cell,wherein the ADCC activity of the anti-CTLA4 antibody is two times orhigher than the ADCC activity of ipilimumab in vitro, and wherein bothantibodies comprise wild type human IgG1 Fc region. In some embodiments,the EC50 of the anti-CTL4 antibody ADCC activity is 50% or less than theEC50 of ipilimumab ADCC activity in vitro. Assays for measuring ADCCactivities are described in Examples 3 and 15. In some embodiments, theanti-CTLA4 antibody depletes Treg cells selectively in tumormicroenvironment (e.g., reducing percentage of Treg cells in tumorinfiltrating lymphocytes), as compared to PBMC or spleen in a mousecancer model. See, e.g., Example 18.

In some embodiments that may be combined with any of the precedingembodiments, the antibody specifically binds to an epitope comprisingamino acid residues at a ligand binding site of human CTLA4, such asCD80 and/or CD86 binding site of human CTLA4. In some embodiments, theantibody specifically binds to an epitope similar to a ligand bindingsite of human CTLA4, such as CD80 and/or CD86 binding site of humanCTLA4. In some embodiments, the antibody specifically binds to anepitope comprising amino acid residues Y105 and L106 of human CTLA4,wherein the numbering of the amino acid residues is according to SEQ IDNO: 207. In some embodiments, the antibody does not bind to residue I108of human CTLA4, wherein the numbering of the amino acid residues isaccording to SEQ ID NO: 207. In some embodiments, the anti-CTLA4antibody blocks binding of CD80 and/or CD86 to human CTLA4. In someembodiments, the anti-CTLA4 antibody has an IC50 higher than the IC50 ofipilimumab for blocking binding of CD80 and/or CD86 to human CTLA4. Insome embodiments, the anti-CTLA4 antibody has an IC50 that is 3.5 timesor higher (including 3.9 times or higher) than the IC50 of ipilimumabfor blocking binding of CD80 and/or CD86 to human CTLA4 in an assay thatCD86 or CD80 is plate bound and CTLA4 is in solution or CTLA4 displayedon cell surface. See Example 13, Table 23, FIGS. 57A-57D and 58. Assaysfor testing antibody's blocking activities (ligand competition) and IC50are described in Examples 3 and 13.

In some embodiments that may be combined with any of the precedingembodiments, the antibody comprises a heavy chain variable region and alight chain variable region, a) wherein the heavy chain variable regioncomprises an HVR-H1, an HVR-H2, and an HVR-H3, wherein the HVR-H1comprises an amino acid sequence according to a formula selected fromthe group consisting of: Formula (I): X1TFSX2YX3IHWV (SEQ ID NO: 1),wherein X1 is F or Y, X2 is D or G, and X3 is A, G, or W; Formula (II):YSIX1SGX2X3WX4WI (SEQ ID NO: 2), wherein X1 is S or T, X2 is H or Y, X3is H or Y, and X4 is A, D, or S; and Formula (III): FSLSTGGVAVX1WI (SEQID NO: 3), wherein X1 is G or S; wherein the HVR-H2 comprises an aminoacid sequence according to a formula selected from the group consistingof: Formula (IV): IGX1IX2HSGSTYYSX3SLKSRV (SEQ ID NO: 4), wherein X1 isD or E, X2 is S or Y, and X3 is P or Q; Formula (V):IGX1ISPSX2GX3TX4YAQKFQGRV (SEQ ID NO: 5), wherein X1 is I or W, X2 is Gor S, X3 is G or S, and X4 is K or N; and Formula (VI):VSX1ISGX2GX3X4TYYADSVKGRF (SEQ ID NO: 6), wherein X1 is A, G, or S, X2is S or Y, X3 is G or S, and X4 is S or T; and wherein the HVR-H3comprises an amino acid sequence according to a formula selected fromthe group consisting of: Formula (VII): ARX1X2X3X4FDX5 (SEQ ID NO: 7),wherein X1 is G, R, or S, X2 is A, I, or Y, X3 is D, V, or Y, X4 is A,E, or Y, and X5 is I or Y; Formula (VIII): ARX1GX2GYFDX3 (SEQ ID NO: 8),wherein X1 is D or L, X2 is F or Y, and X3 is V or Y; Formula (IX):ARX1X2X3X4AX5X6FDY (SEQ ID NO: 9), wherein X1 is L or R, X2 is I or P,X3 is A or Y, X4 is S or T, X5 is T or Y, and X6 is A or Y; Formula (X):ARDX1X2X3GSSGYYX4GFDX5 (SEQ ID NO: 10), wherein X1 is I or V, X2 is A orH, X3 is P or S, X4 is D or Y, and X5 is F or V; and b) wherein thelight chain variable region comprises an HVR-L1, an HVR-L2, and anHVR-L3, wherein the HVR-L1 comprises an amino acid sequence according toa formula selected form the group consisting of: Formula (XI):RASQX1X2X3SX4LX5 (SEQ ID NO: 11), wherein X1 is G or S, X2 is I or V, X3is G or S, X4 is S or Y, and X5 is A or N; Formula (XII):RASQX1VX2X3RX4LA (SEQ ID NO: 12), wherein X1 is S or T, X2 is F, R, orS, X3 is G or S, and X4 is F or Y; and Formula (XIII):RASX1SVDFX2GX3SFLX4 (SEQ ID NO: 13), wherein X1 is E or Q, X2 is D, F,H, or Y, X3 is F, I, or K, and X4 is A, D, or H; wherein the HVR-L2comprises an amino acid sequence according to Formula (XIV):X1ASX2X3X4X5GX6 (SEQ ID NO: 14), wherein X1 is A or D, X2 is N, S, or T,X3 is L or R, X4 is A, E, or Q, X5 is S or T, and X6 is I or V; andwherein the HVR-L3 comprises an amino acid sequence according to aformula selected from the group consisting of: Formula (XV):YCX1X2X3X4X5X6PX7T (SEQ ID NO: 15), wherein X1 is E, Q, or V, X2 is H orQ, X3 is A, G, H, R, or S, X4 is D, L, S, or Y, X5 is E, G, P, Q, or S,X6 is L, T, V, or W, and X7 is F, L, P, W, or Y; Formula (XVI):YCQQX1X2X3WPPWT (SEQ ID NO: 16), wherein X1 is S or Y, X2 is D or Y, andX3 is Q or Y; and Formula (XVII): YCQX1YX2SSPPX3YT (SEQ ID NO: 17),wherein X1 is H or Q, X2 is T or V, and X3 is E or V. In someembodiments, the HVR-H1 comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOS: 18-29, the HVR-H2 comprises an aminoacid sequence selected from the group consisting of SEQ ID NOS: 30-39,the HVR-H3 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 40-52, the HVR-L1 comprises an amino acidsequence selected from the group consisting of SEQ ID NOS: 53-65, theHVR-L2 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 66-69, and the HVR-L3 comprises an amino acidsequence selected from the group consisting of SEQ ID NOS: 70-81. Insome embodiments, the antibody comprises: a) an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 18, an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 30, an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 40, an HVR-L1 comprising the amino acid sequenceof SEQ ID NO: 53, an HVR-L2 comprising the amino acid sequence of SEQ IDNO: 66, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:70; b) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 19, anHVR-H2 comprising the amino acid sequence of SEQ ID NO: 31, an HVR-H3comprising the amino acid sequence of SEQ ID NO: 41, an HVR-L1comprising the amino acid sequence of SEQ ID NO: 54, an HVR-L2comprising the amino acid sequence of SEQ ID NO: 67, and an HVR-L3comprising the amino acid sequence of SEQ ID NO: 71; c) an HVR-H1comprising the amino acid sequence of SEQ ID NO: 20, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 32, an HVR-H3comprising the amino acid sequence of SEQ ID NO: 42, an HVR-L1comprising the amino acid sequence of SEQ ID NO: 55, an HVR-L2comprising the amino acid sequence of SEQ ID NO: 66, and an HVR-L3comprising the amino acid sequence of SEQ ID NO: 72; d) an HVR-H1comprising the amino acid sequence of SEQ ID NO: 21 an HVR-H2 comprisingthe amino acid sequence of SEQ ID NO: 33, an HVR-H3 comprising the aminoacid sequence of SEQ ID NO: 43, an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 56, an HVR-L2 comprising the amino acid sequenceof SEQ ID NO: 68, and an HVR-L3 comprising the amino acid sequence ofSEQ ID NO: 73; e) an HVR-H1 comprising the amino acid sequence of SEQ IDNO: 22, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34,an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 44, an HVR-LLcomprising the amino acid sequence of SEQ ID NO: 57, an HVR-L2comprising the amino acid sequence of SEQ ID NO: 66, and an HVR-L3comprising the amino acid sequence of SEQ ID NO: 74; f) an HVR-H1comprising the amino acid sequence of SEQ ID NO: 23, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 35, an HVR-H3comprising the amino acid sequence of SEQ ID NO: 45, an HVR-L1comprising the amino acid sequence of SEQ ID NO: 58, an HVR-L2comprising the amino acid sequence of SEQ ID NO: 66, and an HVR-L3comprising the amino acid sequence of SEQ ID NO: 75; g) an HVR-H1comprising the amino acid sequence of SEQ ID NO: 24, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 32, an HVR-H3comprising the amino acid sequence of SEQ ID NO: 46, an HVR-L1comprising the amino acid sequence of SEQ ID NO: 59, an HVR-L2comprising the amino acid sequence of SEQ ID NO: 66, and an HVR-L3comprising the amino acid sequence of SEQ ID NO: 76; h) an HVR-H1comprising the amino acid sequence of SEQ ID NO: 25, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 36, an HVR-H3comprising the amino acid sequence of SEQ ID NO: 47, an HVR-L1comprising the amino acid sequence of SEQ ID NO: 60, an HVR-L2comprising the amino acid sequence of SEQ ID NO: 69, and an HVR-L3comprising the amino acid sequence of SEQ ID NO: 77; i) an HVR-H1comprising the amino acid sequence of SEQ ID NO: 26, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 37, an HVR-H3comprising the amino acid sequence of SEQ ID NO: 48, an HVR-L1comprising the amino acid sequence of SEQ ID NO: 61, an HVR-L2comprising the amino acid sequence of SEQ ID NO: 66, and an HVR-L3comprising the amino acid sequence of SEQ ID NO: 78; j) an HVR-H1comprising the amino acid sequence of SEQ ID NO: 27, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 32, an HVR-H3comprising the amino acid sequence of SEQ ID NO: 49, an HVR-L1comprising the amino acid sequence of SEQ ID NO: 62, an HVR-L2comprising the amino acid sequence of SEQ ID NO: 67, and an HVR-L3comprising the amino acid sequence of SEQ ID NO: 79; k) an HVR-H1comprising the amino acid sequence of SEQ ID NO: 28, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 37, an HVR-H3comprising the amino acid sequence of SEQ ID NO: 50, an HVR-L1comprising the amino acid sequence of SEQ ID NO: 63, an HVR-L2comprising the amino acid sequence of SEQ ID NO: 67, and an HVR-L3comprising the amino acid sequence of SEQ ID NO: 80; l) an HVR-H1comprising the amino acid sequence of SEQ ID NO: 18, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 38, an HVR-H3comprising the amino acid sequence of SEQ ID NO: 51, an HVR-L1comprising the amino acid sequence of SEQ ID NO: 64, an HVR-L2comprising the amino acid sequence of SEQ ID NO: 67, and an HVR-L3comprising the amino acid sequence of SEQ ID NO: 81; or m) an HVR-H1comprising the amino acid sequence of SEQ ID NO: 29, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 39, an HVR-H3comprising the amino acid sequence of SEQ ID NO: 52, an HVR-L1comprising the amino acid sequence of SEQ ID NO: 65, an HVR-L2comprising the amino acid sequence of SEQ ID NO: 68, and an HVR-L3comprising the amino acid sequence of SEQ ID NO: 77. In some embodimentsthat may be combined with any of the preceding embodiments, the heavychain variable region comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 82-94, and/or the light chain variableregion comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 95-107. In some embodiments that may becombined with any of the preceding embodiments, the antibody comprises:a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 82 or a variant thereof having at least about 90% (e.g., atleast about 92%, 95%, 98%, 99% or more) sequence identity to the aminoacid sequence of SEQ ID NO: 82, and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 95 or a variant thereofhaving at least about 90% (e.g., at least about 92%, 95%, 98%, 99% ormore) sequence identity to the amino acid sequence of SEQ ID NO: 95; b)a heavy chain variable region comprising the amino acid sequence of SEQID NO: 83 or a variant thereof having at least about 90% (e.g., at leastabout 92%, 95%, 98%, 99% or more) sequence identity to the amino acidsequence of SEQ ID NO: 83, and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 96 or a variant thereof having atleast about 90% (e.g., at least about 92%, 95%, 98%, 99% or more)sequence identity to the amino acid sequence of SEQ ID NO: 96; c) aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 84 or a variant thereof having at least about 90% (e.g., at leastabout 92%, 95%, 98%, 99% or more) sequence identity to the amino acidsequence of SEQ ID NO: 84, and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 97 or a variant thereof having atleast about 90% (e.g., at least about 92%, 95%, 98%, 99% or more)sequence identity to the amino acid sequence of SEQ ID NO: 97; d) aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 85 or a variant thereof having at least about 90% (e.g., at leastabout 92%, 95%, 98%, 99% or more) sequence identity to the amino acidsequence of SEQ ID NO: 85, and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 98 or a variant thereof having atleast about 90% (e.g., at least about 92%, 95%, 98%, 99% or more)sequence identity to the amino acid sequence of SEQ ID NO: 98; e) aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 86 or a variant thereof having at least about 90% (e.g., at leastabout 92%, 95%, 98%, 99% or more) sequence identity to the amino acidsequence of SEQ ID NO: 86, and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 99 or a variant thereof having atleast about 90% (e.g., at least about 92%, 95%, 98%, 99% or more)sequence identity to the amino acid sequence of SEQ ID NO: 99; f) aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 87 or a variant thereof having at least about 90% (e.g., at leastabout 92%, 95%, 98%, 99% or more) sequence identity to the amino acidsequence of SEQ ID NO: 87, and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 100 or a variant thereof having atleast about 90% (e.g., at least about 92%, 95%, 98%, 99% or more)sequence identity to the amino acid sequence of SEQ ID NO: 100; g) aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 88 or a variant thereof having at least about 90% (e.g., at leastabout 92%, 95%, 98%, 99% or more) sequence identity to the amino acidsequence of SEQ ID NO: 88, and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 101 or a variant thereof having atleast about 90% (e.g., at least about 92%, 95%, 98%, 99% or more)sequence identity to the amino acid sequence of SEQ ID NO: 101; h) aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 89 or a variant thereof having at least about 90% (e.g., at leastabout 92%, 95%, 98%, 99% or more) sequence identity to the amino acidsequence of SEQ ID NO: 89, and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 102 or a variant thereof having atleast about 90% (e.g., at least about 92%, 95%, 98%, 99% or more)sequence identity to the amino acid sequence of SEQ ID NO: 102; i) aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 90 or a variant thereof having at least about 90% (e.g., at leastabout 92%, 95%, 98%, 99% or more) sequence identity to the amino acidsequence of SEQ ID NO: 90, and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 103 or a variant thereof having atleast about 90% (e.g., at least about 92%, 95%, 98%, 99% or more)sequence identity to the amino acid sequence of SEQ ID NO: 103; j) aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 91 or a variant thereof having at least about 90% (e.g., at leastabout 92%, 95%, 98%, 99% or more) sequence identity to the amino acidsequence of SEQ ID NO: 91, and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 104 or a variant thereof having atleast about 90% (e.g., at least about 92%, 95%, 98%, 99% or more)sequence identity to the amino acid sequence of SEQ ID NO: 104; k) aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 92 or a variant thereof having at least about 90% (e.g., at leastabout 92%, 95%, 98%, 99% or more) sequence identity to the amino acidsequence of SEQ ID NO: 92, and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 105 or a variant thereof having atleast about 90% (e.g., at least about 92%, 95%, 98%, 99% or more)sequence identity to the amino acid sequence of SEQ ID NO: 105; l) aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 93 or a variant thereof having at least about 90% (e.g., at leastabout 92%, 95%, 98%, 99% or more) sequence identity to the amino acidsequence of SEQ ID NO: 93, and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 106 or a variant thereof having atleast about 90% (e.g., at least about 92%, 95%, 98%, 99% or more)sequence identity to the amino acid sequence of SEQ ID NO: 106; or m) aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 94 or a variant thereof having at least about 90% (e.g., at leastabout 92%, 95%, 98%, 99% or more) sequence identity to the amino acidsequence of SEQ ID NO: 94, and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 107 or a variant thereof having atleast about 90% (e.g., at least about 92%, 95%, 98%, 99% or more)sequence identity to the amino acid sequence of SEQ ID NO: 107.

In some embodiments, the anti-CTLA4 antibody described herein comprisesa heavy chain variable region and a light chain variable region, whereinone, two, three, four, five, or six HVRs of the antibody comprise a HVRsequence shown in Table A. In some embodiments, the anti-CTLA4 antibodycomprises a heave chain variable region comprising an HVR-H1, an HVR-H2,and an HVR-H3, wherein the HVR-H1 comprises the amino acid sequence ofSEQ ID NO: 23, or the HVR-H2 comprises the amino acid sequence of SEQ IDNO: 35, or the HVR-H3 comprises the amino acid sequence of SEQ ID NO:45. In some embodiments, the anti-CTLA4 antibody comprises a light chainvariable region comprising an HVR-L1, an HVR-L2, and an HVR-L3, whereinthe HVR-L1 comprises the amino acid sequence of SEQ ID NO: 58, or theHVR-L2 comprises the antibody comprises the amino acid sequence of SEQID NO: 66, or the HVR-L3 comprises the amino acid sequence of SEQ ID NO:75. In some embodiments, the HVR-H2 of the antibody comprises the aminoacid sequence of SEQ ID NO: 35. In some embodiments, the anti-CTLA4antibody comprises (a) a heavy chain variable region comprising anHVR-H1 comprising the amino acid sequence of SEQ ID NO: 23, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 35, and an HVR-H3comprising the amino acid sequence of SEQ ID NO: 45, and/or a lightchain variable region comprising an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 58, an HVR-L2 comprising the amino acid sequenceof SEQ ID NO: 66, and an HVR-L3 comprising the amino acid sequence ofSEQ ID NO: 75. In some embodiments, one, two, three, four, five or sixof the HVRs of the antibody may comprise one, two or three conservativeamino acid substitutions in the HVRs. In some embodiments, theanti-CTLA4 antibody comprises (b) a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 87 or an amino acidsequence having at least 90% (e.g., 91%, 92%, 93%, 95%, 96%, 97%, 98%,or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 87,and/or a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 100 or an amino acid sequence having at least 90% (e.g.,91%, 92%, 93%, 95%, 96%, 97%, 98%, or 99%) sequence identity to theamino acid sequence of SEQ ID NO: 100.

In some embodiments that may be combined with any of the precedingembodiments, the antibody is an antibody fragment. In some embodiments,the fragment is a Fab, Fab′, Fab′-SH, F(ab′)₂, Fv or scFv fragment. Insome embodiments that may be combined with any of the precedingembodiments, the antibody comprises an IgG1, IgG2, IgG3, or IgG4 Fcregion (such as human IgG1, IgG2, IgG3, or IgG4 Fc region). In someembodiments, the antibody comprising a human IgG1 or a variant that hasenhanced ADCC activity. In some embodiments, the antibody comprises ahuman IgG1 with reduced fucosylation (or non-fucosylated). In someembodiments, the antibody is a human antibody.

Other aspects of the present disclosure relate to an antibody thatcompetes or cross-competes for binding to human CTLA4 with any of theantibodies described herein. Also provided herein are antibodies thatbind to the same epitope and/or essentially the same epitope as any ofthe antibodies described herein.

Other aspects of the present disclosure relate to an activatableantibody comprising: a) a first polypeptide comprising, from N-terminusto C-terminus, a masking moiety (MM), a cleavable moiety (CM), and atarget binding moiety (TBM), wherein the MM comprises an amino acidsequence according to Formula (XVIII): X_(m)CX_(n)CZ_(o) (SEQ ID NO:134), wherein m is from 2-10, n is from 3-10, and o is from 1-10,wherein each X is independently an amino acid selected from the groupconsisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,and Y, and wherein each Z is independently an amino acid selected fromthe group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P; whereinthe MM inhibits the binding of the activatable antibody to human CTLA4when the CM is not cleaved; wherein the CM comprises at least a firstcleavage site; and wherein the TBM comprises an antibody heavy chainvariable region (VH); and b) a second polypeptide comprising an antibodylight chain variable region (VL); and wherein the activatable antibodybinds to human CTLA4 via the VH and VL when the CM is cleaved. In someembodiments, m is from 3-10.

Other aspects of the present disclosure relate to an activatableantibody comprising: a) a polypeptide comprising, from N-terminus toC-terminus, a masking moiety (MM), a cleavable moiety (CM), and a targetbinding moiety (TBM), wherein the MM comprises an amino acid sequenceaccording to Formula (XVIII): X_(m)CX_(n)CZ_(o) (SEQ ID NO: 134),wherein m is from 2-10, n is from 3-10, and o is from 1-10, wherein eachX is independently an amino acid selected from the group consisting ofA, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, andwherein each Z is independently an amino acid selected from the groupconsisting of D, A, Y, S, T, N, I, L, F, V, H, and P; wherein the MMinhibits the binding of the activatable antibody to human CTLA4 when theCM is not cleaved; wherein the CM comprises at least a first cleavagesite; and wherein the TBM comprises an antibody light chain variableregion (VL); and b) a second polypeptide comprising an antibody heavychain variable region (VH); and wherein the activatable antibody bindsto human CTLA4 via the VH and VL when the CM is cleaved. In someembodiments, m is from 3-10.

Other aspects of the present disclosure relate to an activatableantibody comprising: a polypeptide comprising, from N-terminus toC-terminus, a masking moiety (MM), a cleavable moiety (CM), and a targetbinding moiety (TBM), wherein the MM comprises an amino acid sequenceaccording to Formula (XVIII): X_(m)CX_(n)CZ_(o) (SEQ ID NO: 134),wherein m is from 2-10, n is from 3-10, and o is from 1-10, wherein eachX is independently an amino acid selected from the group consisting ofA, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, andwherein each Z is independently an amino acid selected from the groupconsisting of D, A, Y, S, T, N, I, L, F, V, H, and P; wherein the MMinhibits the binding of the activatable antibody to human CTLA4 when theCM is not cleaved; wherein the CM comprises at least a first cleavagesite; wherein the TBM comprises from the N-terminus to the C-terminus,an antibody light chain variable region (VL) and an antibody heavy chainvariable region (VH); and wherein the activatable antibody binds tohuman CTLA4 via the VH and VL when the CM is cleaved. In someembodiments, m is from 3-10.

Other aspects of the present disclosure relate to an activatableantibody comprising: a polypeptide comprising, from N-terminus toC-terminus, a masking moiety (MM), a cleavable moiety (CM), and a targetbinding moiety (TBM), wherein the MM comprises an amino acid sequenceaccording to Formula (XVIII): X_(m)CX_(n)CZ_(o) (SEQ ID NO: 134),wherein m is from 2-10, n is from 3-10, and o is from 1-10, wherein eachX is independently an amino acid selected from the group consisting ofA, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, andwherein each Z is independently an amino acid selected from the groupconsisting of D, A, Y, S, T, N, I, L, F, V, H, and P; wherein the MMinhibits the binding of the activatable antibody to human CTLA4 when theCM is not cleaved; wherein the CM comprises at least a first cleavagesite; wherein the TBM comprises from the N-terminus to the C-terminus,an antibody heavy chain variable region (VH) and an antibody light chainvariable region (VL); and wherein the activatable antibody binds tohuman CTLA4 via the VH and VL when the CM is cleaved.

In some embodiments according to any one of the activatable antibodiesdescribed above, m is 2, 3, 4, 5, or 6. In some embodiments, m is 6. Insome embodiments, n is from 6-8. In some embodiments, n is 6. In someembodiments, o is from 1-2. In some embodiments, o is 2. In someembodiments that may be combined with any of the preceding embodiments,each X is not M, W, or C. In some embodiments that may be combined withany of the preceding embodiments, each X in X_(m) of Formula (XVIII) isindependently an amino acid selected from the group consisting of D, A,Y, S, T, N, I, L, F, V, H, and P. In some embodiments that may becombined with any of the preceding embodiments, each X in X_(n) ofFormula (XVIII) is independently an amino acid selected from the groupconsisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In someembodiments, the MM comprises an amino acid sequence selected from thegroup consisting of X_(m)CPDHPYPCXX (SEQ ID NO:181), X_(m)CDAFYPYCXX(SEQ ID NO:182), X_(m)CDSHYPYCXX (SEQ ID NO:183), and X_(m)CVPYYYACXX(SEQ ID NO:184), where m is from 2-10, and where each X is independentlyan amino acid selected from the group consisting of A, C, D, E, F, G, H,I, K, L, M, N, P, Q, R, S, T, V, W, and Y. In some embodiments, each Xis not M, W, or C. In some embodiments, each X is independently an aminoacid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V,H, and P. In some embodiments that may be combined with any of thepreceding embodiments, the masking moiety (MM) comprises an amino acidsequence selected from SEQ ID NOS: 141-147. In some embodiments that maybe combined with any of the preceding embodiments, the MM furthercomprises, at its N-terminus, an additional amino acid sequence. In someembodiments, the additional amino acid sequence comprises the amino acidsequence of SEQ ID NO: 148.

In some embodiments that may be combined with any of the precedingembodiments, the first cleavage site is a protease cleavage site for aprotease selected from the group consisting of urokinase-typeplasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2,MMP-3, MMP-8, MMP-9, MMP-14, Tobacco Etch Virus (TEV) protease, plasmin,Thrombin, Factor X, PSA, PSMA, Cathepsin D, Cathepsin K, Cathepsin S,ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4,Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10,Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE. In someembodiments that may be combined with any of the preceding embodiments,the CM further comprises a first linker (L₁) C-terminal to the firstcleavage site. In some embodiments, the L₁ comprises an amino acidsequence selected from the group consisting of SEQ ID NOS: 156-163. Insome embodiments that may be combined with any of the precedingembodiments, the CM further comprises a second cleavage site. In someembodiments, the second cleavage site is C-terminal to the L₁. In someembodiments, the second cleavage site is a protease cleavage site for aprotease selected from the group consisting of urokinase-typeplasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2,MMP-3, MMP-8, MMP-9, MMP-14, Tobacco Etch Virus (TEV) protease, plasmin,Thrombin, Factor X, PSA, PSMA, Cathepsin D, Cathepsin K, Cathepsin S,ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4,Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10,Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE. In someembodiments, the first and second cleavage sites are different. In someembodiments that may be combined with any of the preceding embodiments,the CM further comprises a second linker (L₂) C-terminal to the secondcleavage site. In some embodiments, the L₂ comprises an amino acidsequence selected from the group consisting of SEQ ID NOS: 156-163. Insome embodiments that may be combined with any of the precedingembodiments, the CM further comprises a third linker (L₃) N-terminal tothe first cleavage site. In some embodiments that may be combined withany of the preceding embodiments, the CM comprises at least a firstprotease cleavage site and is cleaved with one or more proteasesselected from the group consisting of urokinase-type plasminogenactivator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3,MMP-8, MMP-9, MMP-14, Tobacco Etch Virus (TEV) protease, plasmin,Thrombin, Factor X, PSA, PSMA, Cathepsin D, Cathepsin K, Cathepsin S,ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4,Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10,Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE.

In some embodiments that may be combined with any of the precedingembodiments, the activatable antibody comprises a masking moiety (MM)and cleavable moiety (CM) comprising an amino acid sequence according toFormula (XXIX):EVGSYX1X2X3X4X5X6CX7X8X9X10X11X12CX13X14SGRSAGGGGTENLYFQGSGGS (SEQ IDNO: 164), wherein X1 is A, D, I, N, P, or Y, X2 is A, F, N, S, or V, X3is A, H, L, P, S, V, or Y, X4 is A, H, S, or Y, X5 is A, D, P, S, V, orY, X6 is A, D, L, S, or Y, X7 is D, P, or V, X8 is A, D, H, P, S, or T,X9 is A, D, F, H, P, or Y, X10 is L, P, or Y, X11 is F, P, or Y, X12 isA, P, S, or Y, X13 is A, D, N, S, T, or Y, and X14 is A, S, or Y. Insome embodiments that may be combined with any of the precedingembodiments, the activatable antibody comprises an amino acid sequenceselected from the group consisting of SEQ ID NOS: 165-179.

In some embodiments that may be combined with any of the precedingembodiments, the VL comprises an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 58, an HVR-L2 comprising the amino acid sequenceof SEQ ID NO: 66, and an HVR-L3 comprising the amino acid sequence ofSEQ ID NO: 75. In some embodiments that may be combined with any of thepreceding embodiments, the VL comprises the amino acid sequence of SEQID NO: 100, or a variant thereof having at least about 90% (e.g., atleast about 92%, 95%, 98%, 99% or more) sequence identity to the aminoacid sequence of SEQ ID NO: 100. In some embodiments that may becombined with any of the preceding embodiments, the VH comprises anHVR-H1 comprising the amino acid sequence of SEQ ID NO: 23, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 35, and an HVR-H3comprising the amino acid sequence of SEQ ID NO: 45. In some embodimentsthat may be combined with any of the preceding embodiments, the VHcomprises the amino acid sequence of SEQ ID NO: 87, or a variant thereofhaving at least about 90% (e.g., at least about 92%, 95%, 98%, 99% ormore) sequence identity to the amino acid sequence of SEQ ID NO: 87.

Other aspects of the present disclosure relate to a pharmaceuticalcomposition comprising any of the antibodies and/or activatableantibodies described herein and a pharmaceutically acceptable carrier.

Other aspects of the present disclosure relate to a polynucleotideencoding any of the antibodies and/or activatable antibodies describedherein. In some embodiments, the polynucleotide comprises a sequenceselected from SEQ ID NOS: 108-133.

Other aspects of the present disclosure relate to a vector comprisingany of the polynucleotides described herein. In some embodiments, thevector is an expression vector and/or a display vector.

Other aspects of the present disclosure relate to a host cell comprisingany of the polynucleotides and/or vectors described herein. In someembodiments, the host cell is a eukaryotic cell. In some embodiments,the host cell is a Chinese Hamster Ovary (CHO) cell.

Other aspects of the present disclosure relate to a method of making anantibody or activatable antibody comprising culturing any of the hostcells described herein under conditions suitable for producing theantibody or activatable antibody. In some embodiments, the methodfurther comprises recovering the antibody or activatable antibodyproduced by the cell.

Other aspects of the present disclosure relate to a method of treatingor delaying progression of cancer in a subject in need thereofcomprising administering to the subject an effective amount of any ofthe antibodies, activatable antibodies, and/or pharmaceuticalcompositions descried herein. In some embodiments, the cancer is livercancer, a cancer of the digestive system (e.g., colon cancer, colorectalcancer), lung cancer, bone cancer, heart cancer, brain cancer, kidneycancer, bladder cancer, a hematological cancer (e.g., leukemia), skincancer, breast cancer, thyroid cancer, pancreatic cancer, a head and/orneck cancer, an eye-related cancer, a male reproductive system cancer(e.g., prostate cancer, testicular cancer), or a female reproductivesystem cancer (e.g., uterine cancer, cervical cancer). Other aspects ofthe present disclosure relate to a method of reducing size of a solidtumor in a subject in need thereof, wherein the solid tumor has a sizeof about 400-1000 mm³, the method comprises comprising administering tothe subject an effective amount of any of the antibodies, activatableantibodies, and/or pharmaceutical compositions descried herein. In someembodiments, the solid tumor has a size of about 400-800 mm³. In someembodiments, the method further comprises administering to the subjectan effective amount of at least one additional therapeutic agent. Insome embodiments, the at least one additional therapeutic agent isselected from the group consisting of viral gene therapy, immunecheckpoint inhibitors, target therapies, radiation therapies,vaccination therapies, and chemotherapies. In some embodiments, the atleast one additional therapeutic agent is selected from the groupconsisting of pomalyst, revlimid, lenalidomide, pomalidomide,thalidomide, a DNA-alkylating platinum-containing derivative, cisplatin,5-fluorouracil, cyclophosphamide, an anti-CD137 antibody, an anti-PD-1antibody, an anti-PD-L1 antibody, an anti-CD20 antibody, an anti-CD40antibody, an anti-DR5 antibody, an anti-CD1d antibody, an anti-TIM3antibody, an anti-SLAMF7 antibody, an anti-KIR receptor antibody, ananti-OX40 antibody, an anti-HER2 antibody, an anti-ErbB-2 antibody, ananti-EGFR antibody, cetuximab, rituximab, trastuzumab, pembrolizumab,radiotherapy, single dose radiation, fractionated radiation, focalradiation, whole organ radiation, IL-12, IFNα, GM-CSF, a chimericantigen receptor, adoptively transferred T cells, an anti-cancervaccine, and an oncolytic virus. In some embodiments, the methodcomprises administering to the subject an effective amount of theanti-CTLA4 antibody, the activatable antibody or the pharmaceuticalcomposition described herein prior to a surgery or after a surgery toremove the tumor in the subject. In some embodiments, the anti-CD137antibody comprises an antibody heavy chain variable region comprising anHVR-H1 comprising the amino acid sequence FSLSTGGVGVGWI (SEQ ID NO:223), an HVR-H2 comprising the amino acid sequence LALIDWADDKYYSPSLKSRL(SEQ ID NO:224), and an HVR-H3 comprising the amino acid sequenceARGGSDTVIGDWFAY (SEQ ID NO: 225), and an antibody light chain variableregion comprising an HVR-L1 comprising the amino acid sequenceRASQSIGSYLA (SEQ ID NO: 226), an HVR-L2 comprising the amino acidsequence DASNLETGV (SEQ ID NO: 227), and an HVR-L3 comprising the aminoacid sequence YCQQGYYLWT (SEQ ID NO: 228). In some embodiments, theanti-CD137 antibody comprises an antibody heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 229 or a sequencehaving at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99%) to the sequence of SEQ ID NO: 229; and/or an antibody light chainvariable region comprising the amino acid sequence of SEQ ID NO: 230 ora sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99%) to the sequence of SEQ ID NO: 230.

(SEQ ID NO: 229) EVQLVESGGGLVQPGGSLRLSCAASGFSLSTGGVGVGWIRQAPGKGLEWLALIDWADDKYYSPSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARGGSDTVIGDWFMYWGQGTLVTVSS (SEQ ID NO: 230)DIQLTQSPSSLSASVGDRVTITCRASQSIGSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYYLWTFGQG TKVEIK

It is to be understood that one, some, or all of the properties of thevarious embodiments described above and herein may be combined to formother embodiments of the present disclosure. These and other aspects ofthe present disclosure will become apparent to one of skill in the art.These and other embodiments of the present disclosure are furtherdescribed by the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show antibody binding to CTLA4, as determined by ELISA. FIG.1A shows binding of the indicated antibodies to human CTLA4. FIG. 1Bshows binding of the indicated antibodies to canine CTLA4.

FIG. 2 shows species cross-reactivity of the indicated antibodies,isotype control, or vehicle (PBSA) to HEK293F cells transientlyoverexpressing empty vector (pIRES), or mouse or human CTLA4, asdetermined by flow cytometry.

FIG. 3 shows binding of antibody TY21580, or isotype control, toactivated human, monkey, and mouse T cells, as determined by flowcytometry.

FIGS. 4A-4C show antibody specificity for CTLA4, as determined by flowcytometry. FIG. 4A shows binding of the indicated antibodies, or isotypecontrol, to HEK293F cells transiently overexpressing human PD-1, CTLA4,LAG3, TIM3, B7-H3, or empty vector (293F). FIG. 4B shows binding of theindicated antibodies, isotype control, or vehicle (PBSA) to HEK293Fcells transiently overexpressing human CD95, CD120a, OX40, CD40, CTLA4,or empty vector (pIRES). FIG. 4C shows binding of the indicatedantibodies, isotype control, or vehicle (PBSA) to HEK293F cellstransiently overexpressing human TIM3, CTLA4, PD-L1, LAG3, BTLA, VISTA,PD-L2, ICOS, B7-H4, PD-1, B7-H3, or empty vector (pIRES).

FIGS. 5A-5D show the blocking capabilities of the antibodies, asdetermined by ELISA. FIG. 5A shows the ability of antibodies TY21687,TY21689, TY21680, and TY21691 to block human CD80 binding to humanCTLA4. FIG. 5B shows the ability of antibodies TAC2114, TY21585,TY21587, TY21588, TY21589, TY21580, and TY21591 to block human CD80binding to human CTLA4. FIG. 5C shows the ability of antibodies TY21687,TY21689, TY21680, and TY21691 to block human CD86 binding to humanCTLA4. FIG. 5D shows the ability of antibodies TAC2114, TY21585,TY21587, TY21588, TY21589, TY21580, and TY21591 to block human CD86binding to human CTLA4.

FIGS. 6A-6B show the blocking capabilities of the antibodies, asdetermined by FACS. FIG. 6A shows the ability of the indicatedantibodies, isotype control, or vehicle (PBSA) to block human CD80binding to HEK293F cells transiently overexpressing human CTLA4. FIG. 6Bshows the ability of the indicated antibodies, isotype control, orvehicle (PBSA) to block human CD86 binding to HEK293F cells transientlyoverexpressing human CTLA4.

FIG. 7 shows the ability of the indicated antibodies to bind FcRn, asdetermined by surface plasmon resonance (SPR).

FIG. 8A-8B show human peripheral blood mononuclear cell (PBMC)activation by antibody TY21580 or isotype control, as measured by ELISA.FIG. 8A shows the effect on IL-2 secretion from CD3-stimulated humanPBMCs treated with antibody TY21580 or isotype control. FIG. 8B showsthe effect on IFNγ secretion from CD3-stimulated human PBMCs treatedwith antibody TY21580 or isotype control.

FIG. 9 shows the effect on IL-2 secretion from human PBMCs treated withantibody TY21580 in the presence or absence of an anti-CD3 antibody, asmeasured by ELISA.

FIG. 10 shows the effect on IFNγ secretion from human dendritic cells(DCs) co-cultured with allogenic CD4⁺ T cells treated with antibodyTY21580, isotype control, or an anti-PD-1 antibody, as measured byELISA.

FIGS. 11A-11B show the antibody-dependent cell-mediated cytotoxicity(ADCC) activity of exemplary antibodies on HEK293F cells transientlyoverexpressing human CTLA4, as determined by lactate dehydrogenase (LDH)release assay. FIG. 11A shows the ADCC activity of antibody TY21580, orisotype control, on HEK293F cells transiently overexpressing human CTLA4and incubated with human natural killer (NK) cells. FIG. 11B shows theADCC activity of antibody TY21580, TAC2114, or isotype control onHEK293F cells transiently overexpressing human CTLA4 and incubated withhuman NK cells.

FIGS. 12A-12B show the ADCC activity of exemplary antibodies on humanTregs isolated from two donors, as determined by calcein-AM releaseassay. FIG. 12A shows the ADCC activity of antibody TY21580, TAC2114, orisotype control on human Treg cells (from donor #96) incubated withhuman NK cells. FIG. 12B shows the ADCC activity of antibody TY21580,TAC2114, or isotype control on human Treg cells (from donor #12)incubated with human NK cells.

FIG. 13 shows the complement-dependent cytotoxicity (CDC) activity ofantibody TY21580, or isotype control, on HEK293F cells transientlyoverexpressing human CTLA4, as determined by calcein-AM release assay.

FIG. 14 shows the CDC activity of antibody TY21580, or isotype control,on activated human CD4⁺ T cells, as determined by calcein-AM releaseassay.

FIGS. 15A-15C show the in vivo anti-tumor efficacy of antibody TY21580,or isotype control, in an MC38 syngeneic mouse colorectal tumor model.FIG. 15A shows the tumor growth curves of different treatment groups offemale C57BL/6 mice bearing MC38-established tumors. Data pointsrepresent group mean; error bars represent SEM. FIG. 15B showsindividual tumor growth curves for each group tested. FIG. 15C showsre-challenge studies indicating the long lasting memory of immunityagainst MC38 tumor cells.

FIG. 16 shows the in vivo anti-tumor efficacy of antibody TY21580, orisotype control, in a CT26 syngeneic mouse colorectal tumor model. Tumorgrowth curves of different treatment groups of female C57BL/6 micebearing CT26-established tumors are shown. Data points represent groupmean; error bars represent SEM.

FIG. 17 shows the in vivo anti-tumor efficacy of antibodies TY21586,TY21580, or isotype control in an H22 syngeneic mouse liver tumor model.Tumor growth curves of different treatment groups of female C57BL/6 micebearing H22-established tumors are shown. Data points represent groupmean; error bars represent SEM.

FIG. 18 shows the in vivo anti-tumor efficacy of antibodies TY21580,TY21687, TY21687, TY21691 and TY21580, or isotype control in a Lewissyngeneic mouse lung tumor model. Tumor growth curves of differenttreatment groups of female C57BL/6 mice bearing Lewis-established tumorsare shown. Data points represent group mean; error bars represent SEM.

FIG. 19 shows the in vivo anti-tumor efficacy of antibody TY21580, orisotype control, in a PAN02 syngeneic mouse pancreatic tumor model.Tumor growth curves of different treatment groups of female C57BL/6 micebearing PAN02-established tumors are shown. Data points represent groupmean; error bars represent SEM.

FIGS. 20A-20B show the in vivo anti-tumor efficacy of a monotherapy ofantibody TY21580, an anti-CD137 antibody, or isotype control, as well asan TY21580+anti-CD137 combination therapy, in a 3LL syngeneic mouse lungtumor model. FIG. 20A shows the tumor growth curves of differenttreatment groups of female C57BL/6 mice bearing 3LL-established tumors.Data points represent group mean; error bars represent SEM. FIG. 20Bshows individual tumor growth curves for each group tested.

FIG. 21 shows a re-challenge study indicating the long lasting memory ofimmunity against H22 mouse liver tumor cells. Mice with a completeresponse in an TY21580 treatment group were subcutaneously re-challengedwith H22 tumor cells at the opposite flank on Day 59. Naïve mice werealso inoculated with H22 tumor cells at the same time.

FIG. 22 shows a time course of the blood concentrations of the indicatedantibodies intravenously administered at a concentration of 10 mg/kg tofemale BALB/c mice, as determined by ELISA.

FIG. 23 shows a time course of the blood concentrations of the indicatedantibodies intravenously administered at a concentration of 10 mg/kg incynomolgus monkeys, as determined by ELISA.

FIG. 24 shows a time course of the blood concentrations of the indicatedantibodies intravenously administered at a concentration of 10 mg/kg incynomolgus monkeys, in comparison to the appearance of anti-drugantibodies (ADAs) in these monkeys, as determined by ELISA.

FIGS. 25A-25B show the average spleen weight of male and female BALB/cmice after repeat intraperitoneal administration of either antibodyTY21580 or vehicle control. FIG. 25A shows the average spleen weight ofmale BALB/c mice after repeat intraperitoneal administration of eitherantibody TY21580 or vehicle control on days 1, 4, 7, and 11. FIG. 25Bshows the average spleen weight of female BALB/c mice after repeatintraperitoneal administration of either antibody TY21580 or vehiclecontrol on days 1, 4, 7, and 11.

FIG. 26 shows the histopathology of BALB/c mice after repeatintraperitoneal administration of either antibody TY21580 or vehiclecontrol on days 1, 4, 7, and 11.

FIGS. 27A-27B shows the stability of exemplary antibodies after storageat high concentration. FIG. 27A shows the size exclusion chromatography(SEC) profile of antibody TY21586 after storage at >100 mg/mL. FIG. 27Bshows the SEC profile of antibody TY21580 after storage at >100 mg/mL.

FIG. 28 shows the SEC profiles of exemplary antibodies under acceleratedstress conditions.

FIG. 29 shows a schematic of the selection process for self-blockingpeptides using the Fab fragment of the anti-CTLA4 antibody displayed onyeast surface.

FIG. 30 shows a schematic of the selection process for self-blockingpeptides using the scFv fragment of the anti-CTLA4 antibody displayed onyeast surface.

FIGS. 31A-31B show functional display of Fabs and scFvs targeting CTLA4on yeast, as determined by flow cytometry. FIG. 31A shows functionaldisplay of Fabs targeting CTLA4 on the surface of yeast. FIG. 31B showsfunctional display of scFvs targeting CTLA4 on the surface of yeast.

FIG. 32 shows an exemplary selection process for activatable antibodiestargeting human CTLA4. A yeast library displaying fusion proteins weresubjected to several rounds of FACS-based screening.

FIGS. 33A-33B show CTLA4 binding affinity of exemplary CTLA4 activatableantibody clones, as determined by flow cytometry. FIG. 33A shows bindingaffinity of CTLA4 activatable antibody clones in the scFv format,including the CTLA4 activatable antibody clone B13287 with the maskingpeptide intact, or with the masking peptide cleaved by the TEV protease,as compared to the scFv fragment of the target antibody with no maskingpeptide. FIG. 33B shows CTLA4 binding affinity of CTLA4 activatableantibody clones in the Fab format, including the CTLA4 activatableantibody clone B13189 with the masking peptide intact, or with themasking peptide cleaved by the TEV protease, as compared to the Fabfragment of the target antibody with no masking peptide.

FIGS. 34A-34B show the masking efficiency of exemplary CTLA4 activatableantibodies TY22401, TY22403, TY22402, and TY22404, as compared to theparental antibody TY21580. FIG. 34A shows the association anddissociation curves of the indicated activatable antibodies as comparedto the parental antibody TY21580, as determined by the ForteBio system.FIG. 34B shows a graph of the relative ratio of bound activatableantibodies, as compared to the parental antibody TY21580.

FIGS. 35A-35B show the masking efficiency of exemplary CTLA4 activatableantibodies against recombinant human CTLA4-Fc, as determined by ELISA.FIG. 35A shows a first batch of ELISA data indicating binding of CTLA4activatable antibodies TY22401, TY22402, TY22403, TY22404 to recombinanthuman CTLA4-Fc, as compared to the parental antibody TY21580. FIG. 35Bshows binding of CTLA4 activatable antibodies TY22563, TY22564, TY22565,TY22566 to recombinant human CTLA4-Fc, as compared to the parentalantibody TY21580.

FIGS. 36A-36B show activity of CTLA4 activatable antibody TY22404 uponremoval of the masking peptide. FIG. 36A shows SDS-PAGE results ofactivatable antibody TY22404 with no treatment, treated with theprotease uPA, or treated with 5 or 10 units of the protease MMP-9. FIG.36B shows binding of activatable antibody TY22404 with no treatment,treated with the protease uPA, or treated with the protease MMP-9, ascompared to the parental antibody TY21580, determined by ELISA.

FIGS. 37A-37C show the size-exclusion chromatography (SEC) profiles ofexemplary activatable antibodies under accelerated stress conditions.FIG. 37A shows the SEC profiles of activatable antibody TY22402 aftersix cycles of freezing and thawing, as compared to the controlcondition. FIG. 37B shows the SEC profiles of activatable antibodyTY22402 after seven days at 50° C., as compared to the controlcondition. FIG. 37C shows the percentages of SEC main peak area of theexemplary activatable antibodies after seven days at 50° C., afterstorage at 40° C. for up to 28 days, or after six cycles of freezing andthawing, as compared to the control condition.

FIG. 38 shows the percentages of SEC main peak area of activatableantibodies TY22401 and TY22402 after storage at approximately 8 mg/mL orat >150 mg/mL.

FIG. 39 shows the masking efficiency of untreated activatable antibodiesTY21580, TY22401, TY22402 and TY22566 incubated at pH 3.7 for 30minutes, or incubated at pH 3.7 for an hour, as determined by theForteBio System.

FIGS. 40A-40B show human peripheral blood mononuclear cell (PBMC)activation by isotype control antibody, parental antibody TY21580, orexemplary CTLA4 activatable antibodies TY22401, TY22402, or TY22404, asmeasured by ELISA. FIG. 40A shows the effect on IL-2 secretion fromCD3-primed human PBMCs stimulated with isotype control antibody,parental antibody TY21580, and exemplary CTLA4 activatable antibodiesTY22401, TY22402, or TY22404. FIG. 40B shows the effect on IFNγsecretion from CD3-primed human PBMCs stimulated with isotype controlantibody, parental antibody TY21580, and exemplary CTLA4 activatableantibodies TY22401, TY22402, or TY22404.

FIG. 41 shows the antibody-dependent cell-mediated cytotoxicity (ADCC)activity of isotype control antibody, the parental antibody TY21580, orexemplary activatable antibodies TY22401, TY21580, or TY22404 on HEK293Fcells transiently overexpressing human CTLA4, as determined by an ADCCreporter gene assay.

FIGS. 42A-42B show the in vivo anti-tumor efficacy of parental antibodyTY21580, isotype control antibody, or exemplary CTLA4 activatableantibodies TY22401, TY22402, or TY22566 in an MC38 syngeneic mousecolorectal tumor model. FIG. 42A shows the tumor growth curves ofdifferent treatment groups of female C57BL/6 mice bearingMC38-established tumors. Data points represent group mean; error barsrepresent SEM. FIG. 42B shows individual tumor growth curves for thegroups treated with TY21580, TY22401, TY22402, and TY22566.

FIG. 43 shows the in vivo anti-tumor efficacy of isotype controlantibody, parental antibody TY21580, or one of three activatableantibodies, in a CT26 syngeneic mouse colorectal tumor model. Tumorgrowth curves of different treatment groups of female C57BL/6 micebearing CT26-established tumors are shown. Data points represent groupmean; error bars represent SEM.

FIG. 44 shows the in vivo anti-tumor efficacy of isotype controlantibody, parental antibody TY21580, or one of three activatableantibodies, in an H22 syngeneic mouse liver tumor model. Tumor growthcurves of different treatment groups of female C57BL/6 mice bearingH22-established tumors are shown. Data points represent group mean;error bars represent SEM.

FIGS. 45A-45B show the in vivo anti-tumor efficacy of parental antibodyTY21580, isotype control antibody, and exemplary activatable antibodiesTY22401, TY22402, or TY22566 in a 3LL syngeneic mouse lung tumor model.FIG. 45A shows the tumor growth curves of different treatment groups offemale C57BL/6 mice bearing 3LL-established tumors. Data pointsrepresent group mean; error bars represent SEM. FIG. 45B showsindividual tumor growth curves for the groups treated with TY21580,TY22401, TY22402, and TY22566.

FIGS. 46A-46C show time courses of the blood concentrations of the testarticles (TAs) intravenously administered at a concentration of 10 mg/kgto female BALB/c mice, as determined by ELISA. FIG. 46A shows a timecourse of the blood concentrations of the activatable antibody TY22401intravenously administered at a concentration of 10 mg/kg to femaleBALB/c mice, as compared to the parental antibody TY21580. FIG. 46Bshows a time course of the blood concentrations of the activatableantibody TY22402 intravenously administered at a concentration of 10mg/kg to female BALB/c mice, as compared to the parental antibodyTY21580. FIG. 46C shows a time course of the blood concentrations of theactivatable antibody TY22404 intravenously administered at aconcentration of 10 mg/kg to female BALB/c mice, as compared to theparental antibody TY21580.

FIG. 47 shows the repeated dosing toxicity of isotype control antibody,parental antibody TY21580, and exemplary activatable antibodies TY22566,TY22401, and TY22402 using the NOD mouse model. Percent survival rateover 20 days were shown for each treatment group.

FIGS. 48A-48C show the average spleen weight of BALB/c mice after repeatintraperitoneal administration of the indicated activatable antibodies.FIG. 48A shows the average spleen weight of BALB/c mice after repeatintraperitoneal administration of activatable antibody TY22402, parentalantibody TY21580, or isotype control on days 1, 4, 7, and 11. FIG. 48Bshows the average spleen weight of BALB/c mice after repeatintraperitoneal administration of activatable antibody TY22566, parentalantibody TY21580, or isotype control on days 1, 4, 7, and 11. FIG. 48Cshows the average spleen weight of BALB/c mice after repeatintraperitoneal administration of activatable antibody TY22401, parentalantibody TY21580, or isotype control on days 1, 4, 7, and 11.

FIG. 49 shows the size-exclusion chromatography (SEC) profiles of theindicated activatable antibodies after seven days at 50° C., as comparedto the control condition.

FIG. 50 shows the size-exclusion chromatography (SEC) profiles of theindicated activatable antibodies after storage at 40° C. for 7, 14, 21,or 28 days, as compared to the control condition.

FIG. 51 shows the size-exclusion chromatography (SEC) profiles of theindicated activatable antibodies after six cycles of freezing andthawing, as compared to the control condition.

FIG. 52 shows the percentages of SEC main peak ratios of the indicatedactivatable antibodies after storage at >115 mg/mL.

FIG. 53 shows a summary of the stability data.

FIG. 54 depicts a multiple sequence alignment of a portion of human andmouse CTLA4, with contact residues between human CTLA4 and one of CD80,CD86, or Ipilimumab (based on two crystal structures) mapped. Contactamino acids are shaded in gray, key contact amino acids are in boxes,dimer interface amino acids are indicated with a dot, and amino acidsthat are different between mouse and human CTLA4 are underlined andbolded. Sequences shown are represented by SEQ ID NOs: 203-208, from topto bottom.

FIG. 55A depicts the interaction between human CTLA4 and its ligandCD80. FIG. 55B depicts the interaction between human CTLA4 and itsligand CD86. FIG. 55C depicts the structure alignment between human andmouse CTLA4. Human CTLA4 is colored in black, mouse CTLA4 is colored inwhite.

FIGS. 56A-56E depict results from epitope mapping experiments, showingbinding capacity of TY21580 (FIG. 56A), Ipilimumab (FIG. 56B), humanCD80 (FIG. 56C), human CD86 (FIG. 56D), and mouse CD86 (FIG. 56E) tohuman CTLA4, mouse CTLA4, and CTLA4 mutants by flow cytometry.

FIGS. 57A-57D depict the effect of TY21580 and Ipilimumab onreceptor-ligand binding blockade between human CTLA4 and CD80 or CD86.FIGS. 57A and 57B show binding curves of human CD80 (FIG. 57A) or CD86(FIG. 57B) to plate-bound human recombinant CTLA4 proteins in thepresence of serial dilutions of TY21580, Ipilimumab, or an isotypecontrol antibody, as measured by ELISA. FIGS. 57C and 57D show bindingcurves of human recombinant CTLA4 proteins to plate-bound human CD80(FIG. 57C) or CD86 (FIG. 57D) in the presence of serial dilutions ofTY21580, Ipilimumab, or an isotype control antibody, as measured byELISA.

FIG. 58 depicts CTLA4 blocking-mediated reporter signaling activation ofthe CD28 pathway by anti-CTLA4 antibodies. Jurkat/CTLA4 and aAPC/Rajicells were co-cultured in the presence of serially diluted anti-CTLA4antibodies, with a human IgG1 anti-HEL antibody as an isotype control.Luminescence signals were measured with Bio-Glo luciferase substrateafter overnight incubation and relative luciferase units (RLU) werenormalized against a blank control. Results are expressed as mean RLUfold±SEM. Experiments were performed in triplicate. Note: The data pointfor the top concentration of TY21580 (500 μg/mL) was excluded fromanalysis while fitting the curve as an apparent hook effect was observedat this point.

FIG. 59 depicts ADCC reporter signaling activation by anti-CTLA4antibodies. The Jurkat/NFAT-Luc/CD16 cells and HEK293F/hCTLA4 cells wereco-cultured in the presence of serially diluted anti-CTLA4 antibodies,with a human IgG1 anti-HEL antibody as isotype control. Luminescencesignals were measured with ONE-Glo luciferase substrate after 6 hoursincubation. Relative luciferase units (RLU) were normalized against theblank control, and the results were expressed as mean RLU±SEM.Experiments were performed in triplicate.

FIGS. 60A and 60B depict tumor growth curves of MC38 tumor bearing micetreated with anti-CTLA4 antibodies. FIG. 60A depicts group averagedtumor growth over time in MC38 tumor bearing mice treated with anisotype control antibody (1 mg/kg BIW), TY21580 (1 mg/kg or 0.2 mg/kgBIW), or Ipilimumab (1 mg/kg or 0.2 mg/kg BIW). Data points representmean values; error bars represent standard error of the mean (SEM). FIG.60B depicts tumor growth over time in individual MC38 tumor bearing micetreated with an isotype control antibody (Group-1), TY21580 (Group-2 andGroup-3), or Ipilimumab (Group-4 and Group-5).

FIGS. 61A and 61B depict the effect of TY21580 and Ipilimumab onintra-tumoral regulatory T (Treg) cell levels in subcutaneous MC38tumors from mice treated with TY21580 or Ipilimumab. FIG. 61A shows thepercentages of T regulatory (Treg) cells (CD4+CD25+) in CD4+ T cellsisolated from the tumors. FIG. 61B depicts the ratio of cytotoxic Tlymphocytes (CD8+ T cells) to Treg cells (i.e., the CD8+/Treg ratio) inCD4+ T cell subpopulations isolated from the tumors. Each data pointrepresents the data from one mouse. Statistical analyses were done withPrism 7 (GraphPad Software). P-values were calculated using Multiple Ttest. ns: P>0.05; **: 0.001<P<0.01, ***: P<0.001.

FIGS. 62A and 62B depict the effect of TY21580 and Ipilimumab onintra-tumoral regulatory T (Treg) cell levels in subcutaneous CT26tumors from mice treated with TY21580 or Ipilimumab. FIG. 62A shows thepercentages of T regulatory (Treg) cells (CD4+CD25+) in CD4+ T cellsisolated from the tumors. FIG. 62B depicts the ratio of cytotoxic Tlymphocytes (CD8+ T cells) to Treg cells (i.e., the CD8+/Treg ratio) inCD4+ T cell subpopulations isolated from the tumors. Each data pointrepresents the data from one mouse. Statistical analyses were done withPrism 7 (GraphPad Software). P-values were calculated using Multiple Ttest. ns: P>0.05; **: 0.001<P<0.01, ***: P<0.001.

FIG. 63 depicts CTLA4 expression levels measured by mean fluorescenceintensity (MFI) on FOXP3⁺ CD4⁺ Treg cells in CT26 tumor bearing micetreated with an isotype control antibody or TY21580. Each data pointrepresents the data from one mouse. Statistical analyses were done withPrism 7 (GraphPad Software). P-values were calculated using Multiple Ttest. ns: P>0.05; **: 0.001<P<0.01, ***: P<0.001.

FIGS. 64A-64D depict tumor growth curves of mouse H22 liver cancerbearing mice treated with TY21580 or an isotype control antibody. FIG.64A depicts group averaged tumor growth with TY21580 treatment beginningwhen tumors reached 500 mm³ or 800 mm³, or with isotype control antibodytreatment beginning when tumors reached 500 mm³. Data points representmean values of tumors from 8 mice/group, error bars represent standarderror of the mean (SEM). FIGS. 64B-64D depict individual tumor growth ineach mouse. FIG. 64B depicts tumor growth in mice treated with anisotype control antibody, with treatment beginning when tumors reached500 mm³; FIG. 64C depicts tumor growth in mice treated with TY21580,with treatment beginning when tumors reached 500 mm³; FIG. 64D depictstumor growth in mice treated with TY21580, with treatment beginning whentumors reached 800 mm³.

FIGS. 65A and 65B depict masking efficiencies of exemplary activatableantibodies containing masking peptides of variable lengths, as comparedto the parental antibody TY21580. Masking efficiencies were determinedusing ELISA-based methods. FIGS. 65A and 65B represent two experimentsset up using the same experimental methods to test various activatableanti-CTLA4 antibodies.

FIG. 66 depicts the masking efficiency of exemplary activatableantibodies containing cleavage peptides of varying lengths, as comparedto the parental antibody TY21580. Masking efficiencies were determinedusing ELISA-based methods.

DETAILED DESCRIPTION I. General Techniques

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3d edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Current Protocols inMolecular Biology (F. M. Ausubel, et al. eds., (2003)); the seriesMethods in Enzymology (Academic Press, Inc.): PCR 2: A PracticalApproach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and AnimalCell Culture (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; CellBiology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press;Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Celland Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B.Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbookof Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos,eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds.,1994); Current Protocols in Immunology (J. E. Coligan et al., eds.,1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: A Practical Approach (D. Catty, ed., IRLPress, 1988-1989); Monoclonal Antibodies: A Practical Approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); UsingAntibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold SpringHarbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D.Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principlesand Practice of Oncology (V. T. DeVita et al., eds., J. B. LippincottCompany, 1993).

II. Definitions

Before describing the present disclosure in detail, it is to beunderstood that this present disclosure is not limited to particularcompositions or biological systems, which can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the content clearly dictates otherwise. Thus, forexample, reference to “a molecule” optionally includes a combination oftwo or more such molecules, and the like.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

It is understood that aspects and embodiments of the present disclosuredescribed herein include “comprising,” “consisting,” and “consistingessentially of” aspects and embodiments.

The term “and/or” as used herein a phrase such as “A and/or B” isintended to include both A and B; A or B; A (alone); and B (alone).Likewise, the term “and/or” as used herein a phrase such as “A, B,and/or C” is intended to encompass each of the following embodiments: A,B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C;A (alone); B (alone); and C (alone).

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,gamma-carboxyglutamate, and O-phosphoserine. The term “amino acidanalogs” refers to compounds that have the same basic chemical structureas a naturally occurring amino acid but the C-terminal carboxy group,the N-terminal amino group, or side chain functional group has beenchemically modified to another functional group. The term “amino acidmimetics” refers to chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, but thatfunctions similarly to a naturally occurring amino acid. As used herein,the twenty conventional amino acids and their abbreviations followconventional usage. See e.g., Immunology—A Synthesis (2nd Edition, E. S.Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass.(1991)).

The terms “polypeptide,” “protein,” and “peptide” are usedinterchangeably herein and may refer to polymers of two or more aminoacids.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may comprise modification(s)made after synthesis, such as conjugation to a label. Other types ofmodifications include, for example, “caps,” substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotides(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid or semi-solidsupports. The 5′ and 3′ terminal OH can be phosphorylated or substitutedwith amines or organic capping group moieties of from 1 to 20 carbonatoms. Other hydroxyls may also be derivatized to standard protectinggroups. Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl-, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such asarabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs, and basic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), (O)NR2 (“amidate”), P(O)R,P(O)OR′, CO, or CH2 (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

The term “isolated nucleic acid” refers to a nucleic acid molecule ofgenomic, cDNA, or synthetic origin, or a combination thereof, which isseparated from other nucleic acid molecules present in the naturalsource of the nucleic acid. For example, with regard to genomic DNA, theterm “isolated” includes nucleic acid molecules which are separated fromthe chromosome with which the genomic DNA is naturally associated.Preferably, an “isolated” nucleic acid is free of sequences whichnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid of interest.

The term “antibody” is used herein in the broadest sense andspecifically covers monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies, trispecific antibodies), and antibodyfragments (e.g., Fab, Fab′, Fab′-SH, F(ab′)₂, Fv and/or a single-chainvariable fragment or scFv) so long as they exhibit the desiredbiological activity.

In some embodiments, the term “antibody” refers to an antigen-bindingprotein (i.e., immunoglobulin) having a basic four-polypeptide chainstructure consisting of two identical heavy (H) chains and two identicallight (L) chains. Each L chain is linked to an H chain by one covalentdisulfide bond, while the two H chains are linked to each other by oneor more disulfide bonds depending on the H chain isotype. Each heavychain has, at the N-terminus, a variable region (abbreviated herein asV_(H)) followed by a constant region. The heavy chain constant region iscomprised of three domains, C_(H1), C_(H2) and C_(H3). Each light chainhas, at the N-terminus, a variable region (abbreviated herein as V_(I))followed by a constant region at its other end. The light chain constantregion is comprised of one domain, C_(L). The V_(L) is aligned with theV_(H) and the C_(L) is aligned with the first constant domain of theheavy chain (CH1). The pairing of a V_(H) and V_(L) together forms asingle antigen-binding site. An IgM antibody consists of 5 of the basicheterotetramer units along with an additional polypeptide called Jchain, and therefore contains 10 antigen binding sites, while secretedIgA antibodies can polymerize to form polyvalent assemblages comprising2-5 of the basic 4-chain units along with J chain.

The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed hyper-variable regions (HVR) based onstructural and sequence analysis. HVRs are interspersed with regionsthat are more conserved, termed framework regions (FW) (see e.g., Chenet al. (1999) J. Mol. Biol. (1999) 293, 865-881). Each V_(H) and V_(L)is composed of three HVRs and four FWs, arranged from amino-terminus tocarboxy-terminus in the following order:FW-1_HVR-1_FW-2_HVR-2_FW-3_HVR-3_FW4. Throughout the present disclosure,the three HVRs of the heavy chain are referred to as HVR-H1, HVR-H2, andHVR-H3. Similarly, the three HVRs of the light chain are referred to asHVR-L1, HVR-L2, and HVR-L3.

The variable regions of the heavy and light chains contain a bindingdomain that interacts with an antigen. The constant regions of theantibodies may mediate the binding of the immunoglobulin to host tissuesor factors, including various cells of the immune system (e.g., effectorcells) and the first component (Clq) of the classical complement system.Within light and heavy chains, the variable and constant regions arejoined by a “J” region of about 12 or more amino acids, with the heavychain also including a “D” region of about 10 or more amino acids (seee.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2^(nd) ed. RavenPress, N.Y). (1989)).

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa and lambda, based on the amino acidsequences of their constant domains. Depending on the amino acidsequence of the constant domain of their heavy chains (CH), antibodiescan be assigned to different classes or isotypes. There are five classesof antibodies: IgA, IgD, IgE, IgG, and IgM, having heavy chainsdesignated α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu),respectively. The IgG class of antibody can be further classified intofour subclasses IgG1, IgG2, IgG3, and IgG4 by the gamma heavy chains,Y1-Y4, respectively.

The term “antibody derivative” or “derivative” of an antibody refers toa molecule that is capable of binding to the same antigen (e.g., CTLA4)that the antibody binds to and comprises an amino acid sequence of theantibody linked to an additional molecular entity. The amino acidsequence of the antibody that is contained in the antibody derivativemay be a full-length heavy chain, a full-length light chain, any portionor portions of a full-length heavy chain, any portion or portions of thefull-length light chain of the antibody, any other fragment(s) of anantibody, or the complete antibody. The additional molecular entity maybe a chemical or biological molecule. Examples of additional molecularentities include chemical groups, amino acids, peptides, proteins (suchas enzymes, antibodies), and chemical compounds. The additionalmolecular entity may have any utility, such as for use as a detectionagent, label, marker, pharmaceutical or therapeutic agent. The aminoacid sequence of an antibody may be attached or linked to the additionalmolecular entity by chemical coupling, genetic fusion, noncovalentassociation, or otherwise. The term “antibody derivative” alsoencompasses chimeric antibodies, humanized antibodies, and moleculesthat are derived from modifications of the amino acid sequences of aCTLA4 antibody, such as conservation amino acid substitutions,additions, and insertions.

The term “antigen-binding fragment” or “antigen binding portion” of anantibody refers to one or more portions of an antibody that retain theability to bind to the antigen that the antibody bonds to (e.g., CTLA4).Examples of “antigen-binding fragments” of an antibody include (i) a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L)and C_(H1) domains; (ii) a F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the V_(H) and C_(H1) domains;(iv) a Fv fragment consisting of the V_(L) and V_(H) domains of a singlearm of an antibody, (v) a dAb fragment (Ward et al., Nature 341:544-546(1989)), which consists of a V_(H) domain; and (vi) an isolatedcomplementarity determining region (CDR).

The term “binding molecule” encompasses (1) antibody, (2)antigen-binding fragment of an antibody, and (3) derivative of anantibody, each as defined herein.

The term “CTLA4” is used in the present application, and includes thehuman CTLA4 (e.g., UniProt accession number P16410), as well asvariants, isoforms, and species homologs thereof (e.g., mouse CTLA4(UniProt accession number P09793), rat CTLA4 (UniProt accession numberQ9Z1A7), dog CTLA4 (UniProt accession number Q9XSI1), cynomolgus monkeyCTLA4 (UniProt accession number G7PL88), etc.). Accordingly, a bindingmolecule (e.g., an antibody or activatable antibody), as defined anddisclosed herein, may also bind CTLA4 from species other than human. Inother cases, a binding molecule may be completely specific for the humanCTLA4 and may not exhibit species or other types of cross-reactivity.

The term “CTLA4 antibody” refers to an antibody, as defined herein,capable of binding to human CTLA4.

The term “chimeric antibody” refers to an antibody that comprises aminoacid sequences derived from different animal species, such as thosehaving a variable region derived from a human antibody and a murineimmunoglobulin constant region.

The term “compete for binding” refers to the interaction of twoantibodies in their binding to a binding target. A first antibodycompetes for binding with a second antibody if binding of the firstantibody with its cognate epitope is detectably decreased in thepresence of the second antibody compared to the binding of the firstantibody in the absence of the second antibody. The alternative, wherethe binding of the second antibody to its epitope is also detectablydecreased in the presence of the first antibody, can, but need not, bethe case. That is, a first antibody can inhibit the binding of a secondantibody to its epitope without that second antibody inhibiting thebinding of the first antibody to its respective epitope. However, whereeach antibody detectably inhibits the binding of the other antibody withits cognate epitope, whether to the same, greater, or lesser extent, theantibodies are said to “cross-compete” with each other for binding oftheir respective epitope(s).

The term “epitope” refers to a part of an antigen to which an antibody(or antigen-binding fragment thereof) binds. Epitopes can be formed bothfrom contiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope can include various numbers of aminoacids in a unique spatial conformation. Methods of determining spatialconformation of epitopes include, for example, x-ray crystallography,2-dimensional nuclear magnetic resonance, deuterium and hydrogenexchange in combination with mass spectrometry, or site-directedmutagenesis, or all methods used in combination with computationalmodeling of antigen and its complex structure with its binding antibodyand its variants (see e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, G. E. Morris, Ed. (1996)). Once a desiredepitope of an antigen is determined, antibodies to that epitope can begenerated, e.g., using the techniques described herein. The generationand characterization of antibodies may also elucidate information aboutdesirable epitopes. From this information, it is then possible tocompetitively screen antibodies for binding to the same epitope. Anapproach to achieve this is to conduct cross-competition studies to findantibodies that competitively bind with one another, i.e., theantibodies compete for binding to the antigen. A high throughput processfor “binning” antibodies based upon their cross-competition is describedin PCT Publication No. WO 03/48731.

The term “germline” refers to the nucleotide sequences of the antibodygenes and gene segments as they are passed from parents to offspring viathe germ cells. The germline sequence is distinguished from thenucleotide sequences encoding antibodies in mature B cells which havebeen altered by recombination and hypermutation events during the courseof B cell maturation.

The term “glycosylation sites” refers to amino acid residues which arerecognized by a eukaryotic cell as locations for the attachment of sugarresidues. The amino acids where carbohydrate, such as oligosaccharide,is attached are typically asparagine (N-linkage), serine (O-linkage),and threonine (O-linkage) residues. The specific site of attachment istypically signaled by a sequence of amino acids, referred to herein as a“glycosylation site sequence”. The glycosylation site sequence forN-linked glycosylation is: -Asn-X-Ser- or -Asn-X-Thr-, where X may beany of the conventional amino acids, other than proline. The terms“N-linked” and “O-linked” refer to the chemical group that serves as theattachment site between the sugar molecule and the amino acid residue.N-linked sugars are attached through an amino group; O-linked sugars areattached through a hydroxyl group. The term “glycan occupancy” refers tothe existence of a carbohydrate moiety linked to a glycosylation site(i.e., the glycan site is occupied). Where there are at least twopotential glycosylation sites on a polypeptide, either none (0-glycansite occupancy), one (1-glycan site occupancy) or both (2-glycan siteoccupancy) sites can be occupied by a carbohydrate moiety.

The term “host cell” refers to a cellular system which can be engineeredto generate proteins, protein fragments, or peptides of interest. Hostcells include, without limitation, cultured cells, e.g., mammaliancultured cells derived from rodents (rats, mice, guinea pigs, orhamsters) such as CHO, BHK, NSO, SP2/0, YB2/0; human cells (e.g.,HEK293F cells, HEK293T cells; or human tissues or hybridoma cells, yeastcells, insect cells (e.g., S2 cells), bacterial cells (e.g., E. colicells) and cells comprised within a transgenic animal or culturedtissue. The term encompasses not only the particular subject cell butalso the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not be identical to the parent cell, butare still included within the scope of the term “host cell.”

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

The term “humanized antibody” refers to a chimeric antibody thatcontains amino acid residues derived from human antibody sequences. Ahumanized antibody may contain some or all of the CDRs or HVRs from anon-human animal or synthetic antibody while the framework and constantregions of the antibody contain amino acid residues derived from humanantibody sequences.

The term “illustrative antibody” refers to any one of the antibodiesdescribed in the disclosure and designated as those listed in Tables Aand B, and any antibodies comprising the 6 HVRs and/or the VH and VLs ofthe antibodies listed in Tables A and B. These antibodies may be in anyclass (e.g., IgA, IgD, IgE, IgG, and IgM). Thus, each antibodyidentified above encompasses antibodies in all five classes that havethe same amino acid sequences for the V_(L) and V_(H) regions. Further,the antibodies in the IgG class may be in any subclass (e.g., IgG1 IgG2,IgG3, and IgG4). Thus, each antibody identified above in the IgGsubclass encompasses antibodies in all four subclasses that have thesame amino acid sequences for the V_(L) and V_(H) regions. The aminoacid sequences of the heavy chain constant regions of human antibodiesin the five classes, as well as in the four IgG subclasses, are known inthe art.

An “isolated” antibody or binding molecule (e.g., activatable antibody)is one which has been separated from a component of its naturalenvironment. In some embodiments, an antibody is purified to greaterthan 95% or 99% purity as determined by, for example, electrophoretic(e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis)or chromatographic (e.g., ion exchange or reverse phase HPLC). Forreview of methods for assessment of antibody purity, see e.g., Flatmanet al., J. Chromatogr. B 848:79-87 (2007).

The term “K_(a)” refers to the association rate constant of a particularbinding molecule-antigen interaction, where the term “k_(d)” refers tothe dissociation rate constant of a particular binding molecule-antigeninteraction.

The term “K_(D)” refers to the equilibrium dissociation constant of aparticular antibody-antigen interaction. It is obtained from the ratioof k_(d) to k_(a) (i.e., k_(d)/k_(a)) and is expressed as a molarconcentration (M). K_(D) is used as a measure for the affinity of anantibody's binding to its binding partner. The smaller the K_(D), themore tightly bound the antibody is, or the higher the affinity betweenantibody and the antigen. For example, an antibody with a nanomolar (nM)dissociation constant binds more tightly to a particular antigen than anantibody with a micromolar (μM) dissociation constant. K_(D) values forantibodies can be determined using methods well established in the art.One method for determining the K_(D) of an antibody is by using surfaceplasmon resonance, typically using a biosensor system such as a Biacore®system. For example, an assay procedure using the BIACORE™ system(BIAcore assay) is described in at least Example 3 of the presentdisclosure.

The term “mammal” refers to any animal species of the Mammalia class.Examples of mammals include: humans; laboratory animals such as rats,mice, hamsters, rabbits, non-human primates, and guinea pigs; domesticanimals such as cats, dogs, cattle, sheep, goats, horses, and pigs; andcaptive wild animals such as lions, tigers, elephants, and the like.

The term “prevent” or “preventing,” with reference to a certain diseasecondition in a mammal, refers to preventing or delaying the onset of thedisease, or preventing the manifestation of clinical or subclinicalsymptoms thereof.

As used herein, “sequence identity” between two polypeptide sequencesindicates the percentage of amino acids that are identical between thesequences. The amino acid sequence identity of polypeptides can bedetermined conventionally using known computer programs such as Bestfit,FASTA, or BLAST (see e.g., Pearson, Methods Enzymol. 183:63-98 (1990);Pearson, Methods Mol. Biol. 132:185-219 (2000); Altschul et al., J. Mol.Biol. 215:403-410 (1990); Altschul et al., Nucleic Acids Res.25:3389-3402 (1997)). When using Bestfit or any other sequence alignmentprogram to determine whether a particular sequence is, for instance, 95%identical to a reference amino acid sequence, the parameters are setsuch that the percentage of identity is calculated over the full lengthof the reference amino acid sequence and that gaps in homology of up to5% of the total number of amino acid residues in the reference sequenceare allowed. This aforementioned method in determining the percentage ofidentity between polypeptides is applicable to all proteins, fragments,or variants thereof disclosed herein.

As used herein, the term “binds”, “binds to”, “specifically binds”“specifically binds to” or is “specific for” refers to measurable andreproducible interactions such as binding between a target and anantibody, which is determinative of the presence of the target in thepresence of a heterogeneous population of molecules including biologicalmolecules. For example, an antibody that binds to or specifically bindsto a target (which can be an epitope) is an antibody that binds thistarget with greater affinity, avidity, more readily, and/or with greaterduration than it binds to other targets. In one embodiment, the extentof binding of an antibody to an unrelated target is less than about 10%of the binding of the antibody to the target as measured, e.g., by aradioimmunoassay (RIA). In certain embodiments, an antibody thatspecifically binds to a target has a dissociation constant (Kd) of ≤1μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In certain embodiments, anantibody specifically binds to an epitope on a protein that is conservedamong the protein from different species. In another embodiment,specific binding can include, but does not require exclusive binding.

The term “treat”, “treating”, or “treatment”, with reference to acertain disease condition in a mammal, refers causing a desirable orbeneficial effect in the mammal having the disease condition. Thedesirable or beneficial effect may include reduced frequency or severityof one or more symptoms of the disease (i.e., tumor growth and/ormetastasis, or other effect mediated by the numbers and/or activity ofimmune cells, and the like), or arrest or inhibition of furtherdevelopment of the disease, condition, or disorder. In the context oftreating cancer in a mammal, the desirable or beneficial effect mayinclude inhibition of further growth or spread of cancer cells, death ofcancer cells, inhibition of reoccurrence of cancer, reduction of painassociated with the cancer, or improved survival of the mammal. Theeffect can be either subjective or objective. For example, if the mammalis human, the human may note improved vigor or vitality or decreasedpain as subjective symptoms of improvement or response to therapy.Alternatively, the clinician may notice a decrease in tumor size ortumor burden based on physical exam, laboratory parameters, tumormarkers or radiographic findings. Some laboratory signs that theclinician may observe for response to treatment include normalization oftests, such as white blood cell count, red blood cell count, plateletcount, erythrocyte sedimentation rate, and various enzyme levels.Additionally, the clinician may observe a decrease in a detectable tumormarker. Alternatively, other tests can be used to evaluate objectiveimprovement, such as sonograms, nuclear magnetic resonance testing andpositron emissions testing.

The term “vector” refers to a nucleic acid molecule capable oftransporting a foreign nucleic acid molecule. The foreign nucleic acidmolecule is linked to the vector nucleic acid molecule by a recombinanttechnique, such as ligation or recombination. This allows the foreignnucleic acid molecule to be multiplied, selected, further manipulated orexpressed in a host cell or organism. A vector can be a plasmid, phage,transposon, cosmid, chromosome, virus, or virion. One type of vectorscan be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome(e.g., non-episomal mammalian vectors). Another type of vector iscapable of autonomous replication in a host cell into which it isintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Another specific type ofvector capable of directing the expression of expressible foreignnucleic acids to which they are operatively linked is commonly referredto as “expression vectors.” Expression vectors generally have controlsequences that drive expression of the expressible foreign nucleicacids. Simpler vectors, known as “transcription vectors,” are onlycapable of being transcribed but not translated: they can be replicatedin a target cell but not expressed. The term “vector” encompasses alltypes of vectors regardless of their function. Vectors capable ofdirecting the expression of expressible nucleic acids to which they areoperatively linked are commonly referred to “expression vectors.” Otherexamples of “vectors” may include display vectors (e.g., vectors thatdirect expression and display of an encoded polypeptide on the surfaceof a virus or cell (such as a bacterial cell, yeast cell, insect cell,and/or mammalian cell).

As used herein, a “subject”, “patient”, or “individual” may refer to ahuman or a non-human animal. A “non-human animal” may refer to anyanimal not classified as a human, such as domestic, farm, or zooanimals, sports, pet animals (such as dogs, horses, cats, cows, etc.),as well as animals used in research. Research animals may refer withoutlimitation to nematodes, arthropods, vertebrates, mammals, frogs,rodents (e.g., mice or rats), fish (e.g., zebrafish or pufferfish),birds (e.g., chickens), dogs, cats, and non-human primates (e.g., rhesusmonkeys, cynomolgus monkeys, chimpanzees, etc.). In some embodiments,the subject, patient, or individual is a human.

An “effective amount” refers to at least an amount effective, at dosagesand for periods of time necessary, to achieve one or more desired orindicated effects, including a therapeutic or prophylactic result. Aneffective amount can be provided in one or more administrations. Forpurposes of the present disclosure, an effective amount of antibody,drug, compound, or pharmaceutical composition is an amount sufficient toaccomplish prophylactic or therapeutic treatment either directly orindirectly. As is understood in the clinical context, an effectiveamount of a drug, compound, or pharmaceutical composition may or may notbe achieved in conjunction with another drug, compound, orpharmaceutical composition (e.g., an effective amount as administered asa monotherapy or combination therapy). Thus, an “effective amount” maybe considered in the context of administering one or more therapeuticagents, and a single agent may be considered to be given in an effectiveamount if, in conjunction with one or more other agents, a desirableresult may be or is achieved.

III. Binding Molecules that Bind to Human CTLA4

The present disclosure relates, in part, to isolated binding moleculesthat bind to human CTLA4, including CTLA4 antibodies, antigen-bindingfragments of the CTLA4 antibodies, and derivatives of the CTLA4antibodies. In some embodiments, the binding molecules are any of theantibodies described herein, including antibodies described withreference to specific amino acid sequences of HVRs, variable regions(VL, VH), and light and heavy chains (e.g., IgG1, IgG2, IgG4). In someembodiments, the antibodies are human antibodies. In some embodiments,the antibodies are humanized antibodies and/or chimeric antibodies. Insome embodiments, the present disclosure relates to binding moleculesthat bind to human CTLA4, and have at least one (e.g., at least one, atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, or all nine) of the following functionalproperties: (a) bind to human, cynomolgus monkey, mouse, rat, and/or dogCTLA4 with a K_(D) of 500 nM or less; (b) have antagonist activity onhuman CTLA4; (c) do not bind to human PD-1, PD-L1, PD-L2, LAG3, TIM3,B7-H3, CD95, CD120a, OX40, CD40, BTLA, VISTA, ICOS, and/or B7-H4 atconcentration up to 100 nM; (d) are cross-reactive with monkey, mouse,rat, and/or dog CTLA4; (e) induces ADCC effects (e.g., on Tregs); (f)activates human PBMCs (e.g., stimulates secretion of IL-2 and/or IFNγ);(g) are capable of inhibiting tumor cell growth; (h) have therapeuticeffect on a cancer; and (i) block binding of human CTLA4 to human CD80and/or human CD86. In some embodiments, the anti-CTLA4 antibodiesdescribed herein have lower activity in blocking binding of CD80 and/orCD86 to human CTLA4 as compared to ipilimumab in an assay wherein eitherwhen human CD80 and/or CD86 are immobilized (or plate bound) or whenhuman CTLA4 protein is present on cell surface. See FIGS. 57C and 57Dand 58. In some embodiments, the anti-CTLA4 antibodies described hereindeplete Treg cells selectively in tumor microenvironment as compared toTreg depletions in PBMC or spleen. In some embodiments, the anti-CTLA4antibodies described herein have higher Treg depletion activity in tumormicroenvironment as compared to ipilimumab. See FIGS. 61A-B, 62A-B, and63. Also provided herein are one or more anti-CTLA4 antibodies orantigen-binding fragments that cross-compete for binding to human CTLA4with one or more of the antibodies or antigen-binding fragmentsdescribed herein.

In some embodiments, the antibodies or antigen-binding fragments bind tohuman, cynomolgus monkey, mouse, rat, and/or dog CTLA4 with a K_(D) ofabout 500 nM or less (e.g., about 500 nM or less, about 450 nM or less,about 400 nM or less, about 350 nM or less, about 300 nM or less, about250 nM or less, about 200 nM or less, about 150 nM or less, about 100 nMor less, about 90 nM or less, about 80 nM or less, about 70 nM or less,about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30nM or less, about 25 nM or less, about 20 nM or less, about 10 nM orless, about 1 nM or less, about 0.1 nM or less, etc.) In someembodiments, the antibodies or antigen-binding fragments bind to human,cynomolgus monkey, mouse, rat, and/or dog CTLA4 with a K_(D) of about350 nM or less. In some embodiments, the antibodies or antigen-bindingfragments bind to human CTLA4 with a K_(D) of about 100 nM or less. Insome embodiments, the antibodies or antigen-binding fragments bind tohuman CTLA4 with a K_(D) of about 50 nM or less. In some embodiments,the antibodies or antigen-binding fragments bind to human CTLA4 with aK_(D) of about 10 nM or less. Methods of measuring the K_(D) of anantibody or antigen-binding fragment may be carried out using any methodknown in the art, including for example, by surface plasmon resonance,an ELISA, isothermal titration calorimetry, a filter binding assay, anEMSA, etc. In some embodiments, the K_(D) is measured by surface plasmonresonance or an ELISA (see e.g., Example 3 below).

In some embodiments, the antibodies or antigen-binding fragmentsdescribed herein have antagonist activity on human CTLA4. In someembodiments, the antibodies or antigen-binding fragments repress one ormore activities of human CTLA4 when a cell (e.g., a human cell)expressing human CTLA4 is contacted by the antibody or antigen bindingfragment (e.g., CTLA4 blockade as measured by an increase in a reportergene signal using a CLA4 blockage reporter gene assay).

In some embodiments, the antibodies or antigen-binding fragments arecross-reactive with monkey (e.g., cynomolgus monkey), mouse, rat, and/ordog CTLA4. In some embodiments, the antibodies or antigen-bindingfragments are cross-reactive with monkey CTLA4. In some embodiments, theantibodies or antigen-binding fragments are cross-reactive with mouseCTLA4. In some embodiments, the antibodies or antigen-binding fragmentsare cross-reactive with rat CTLA4. In some embodiments, the antibodiesor antigen-binding fragments are cross-reactive with dog CTLA4. In someembodiments, the antibodies or antigen binding fragments are crossreactive with monkey and mouse CTLA4; monkey and rat CTLA4; monkey anddog CTLA4; mouse and rat CTLA4; mouse and dog CTLA4; rat and dog CTLA4;monkey, mouse, and rat CTLA4; monkey, mouse, and dog CTLA4; monkey, rat,and dog CTLA4; mouse, rat, and dog CTLA4; or monkey, mouse, rat, and dogCTLA4. In some embodiments, the antibodies or antigen binding fragmentsare cross-reactive if the antibodies or antigen-binding fragments bindsto a non-human CTLA4 molecule with a K_(D) less than about 500 nM (e.g.,less than about 1 nM, less than about 10 nM, less than about 25 nM, lessthan about 50 nM, less than about 75 nM, less than about 100 nM, lessthan about 150 nM, less than about 200 nM, less than about 250 nM, lessthan about 300 nM, less than about 350 nM, etc.). Methods of measuringantibody cross-reactivity are known in the art, including, withoutlimitation, surface plasmon resonance, an ELISA, isothermal titrationcalorimetry, a filter binding assay, an EMSA, etc. In some embodiments,the cross-reactivity is measured by ELISA (see e.g., Example 3 below).

In some embodiments, the antibodies induce ADCC effects against a CTLA4expressing cell (e.g., against CTLA4-expressing human cells such asTregs) after the antibody binds to the cell-expressed CTLA4. Methods ofmeasuring ADCC effects (e.g., in vitro methods) are known in the art,including, without limitation, via the methods described in Example 3below. In some embodiments, the antibodies induce ADCC effects by morethan about 10% (e.g., induce ADCC by more than about 10%, more thanabout 15%, more than about 20%, more than about 25%, more than about30%, more than about 35%, more than about 40%, etc.) relative to acontrol (e.g., an isotype control or ipilimumab).

In some embodiments, the antibodies or antigen-binding fragments arecapable of inhibiting tumor cell growth and/or proliferation. In someembodiments, the tumor cell growth and/or proliferation is inhibited byat least about 5% (e.g., at least about 5%, at least about 10%, at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, at leastabout 90%, or at least about 99%) when contacted with the antibodies orantigen-binding fragments relative to corresponding tumor cells notcontacted with the antibodies or antigen-binding fragments (or relativeto corresponding tumor cells contacted with an isotype controlantibody). In some embodiments, the antibodies or antigen-bindingfragments are capable of reducing tumor volume in a subject when thesubject is administered the antibodies or antigen-binding fragments. Insome embodiments, the antibodies or antigen-binding fragments arecapable of reducing tumor volume in a subject by at least about 5%(e.g., at least about 5%, at least about 10%, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, or atleast about 99%) relative to the initial tumor volume in the subject(e.g., prior to administration of the antibodies or antigen-bindingfragments; as compared to a corresponding tumor in a subjectadministered an isotype control antibody). Methods of monitoring tumorcell growth/proliferation, tumor volume, and/or tumor inhibition areknown in the art, including, for example, via the methods described inExample 4 below.

In some embodiments, the antibodies or antigen-binding fragments havetherapeutic effect on a cancer. In some embodiments, the antibodies orantigen-binding fragments reduce one or more signs or symptoms of acancer. In some embodiments, a subject suffering from a cancer goes intopartial or complete remission when administered the antibodies orantigen-binding fragments.

In another aspect, the disclosure provides isolated antibodies thatcompete or cross-compete for binding to human CTLA4 with any of theillustrative antibodies of the disclosure, such as TY21585, TY21586,TY21587, TY21588, TY21589, TY21580, TY21591, TY21686, TY21687, TY21689,TY21680, TY21691, and/or TY21692. In a particular embodiment, thepresent disclosure provides isolated antibodies that compete orcross-compete for binding to the same epitope on the human CTLA4 withany of the illustrative antibodies of the disclosure. The ability of anantibody to compete or cross-compete for binding with another antibodycan be determined using standard binding assays known in the art, suchas BIAcore analysis, ELISA assays, or flow cytometry. For example, onecan allow an illustrative antibody of the disclosure to bind to humanCTLA4 under saturating conditions and then measure the ability of thetest antibody to bind to the CTLA4. If the test antibody is able to bindto the CTLA4 at the same time as the illustrative antibody, then thetest antibody binds to a different epitope as the illustrative antibody.However, if the test antibody is not able to bind to the CTLA4 at thesame time, then the test antibody binds to the same epitope, anoverlapping epitope, or an epitope that is in close proximity to theepitope bound by the illustrative antibody. This experiment can beperformed using various methods, such as ELISA, RIA, FACS or surfaceplasmon resonance.

In some embodiments, the antibodies or antigen-binding fragments blockthe binding between CTLA4 and one or more of its binding partners (e.g.,human CTLA4 and human CD80, human CTLA4 and human CD86). In someembodiments, the antibodies or antigen-binding fragments block thebinding between CTLA4 and its ligand in vitro. In some embodiments, theantibody or antigen-binding fragment has a half maximal inhibitoryconcentration (IC₅₀) of about 500 nM or less (e.g., about 500 nM orless, about 400 nM or less, about 300 nM or less, about 200 nM or less,about 100 nM or less, about 50 nM or less, about 25 nM or less, about 10nM or less, about 1 nM or less, etc.) for blocking binding of CTLA4 toCD80 and/or CD86. In some embodiments, the antibody or antigen-bindingfragment has a half maximal inhibitory concentration (IC₅₀) of about 100nM or less for blocking binding of CTLA4 to CD80 and/or CD86. In someembodiments, the antibody or antigen-binding fragment completely blocksbinding of human CTLA4 to CD80 and/or CD86 when provided at aconcentration of about 100 nM or greater (e.g., about 100 nM or greater,about 500 nM or greater, about 1 μM or greater, about 10 μM or greater,etc.). As used herein, the term “complete blocking” or “completelyblocks” refers to the antibody or antigen-binding fragment's ability toreduce binding between a first protein and a second protein by at leastabout 80% (e.g., at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 99%, etc.). Methods of measuringthe ability of an antibody or antigen-binding fragment to block bindingof a first protein (e.g., human CTLA4) and a second protein (e.g., humanCD80 or human CD86) are known in the art, including, without limitation,via BIAcore analysis, ELISA assays, and flow cytometry (see e.g.,Example 3 below). In some embodiments, the anti-CTLA4 antibodiesdescribed herein have lower activity in blocking ligand binding thanipilimumab.

CTLA4 Antibodies

In some aspects, the present disclosure provides an isolated antibodythat binds to human CTLA4. In some embodiments, the antibody binds humanCTLA4 with a K_(D) of 1000 nM or less (e.g., 50 nM or less, 10 nM orless) as measured by surface plasmon resonance. In some embodiments, theantibody is cross-reactive with at least one non-human species selectedfrom cynomolgus monkey, mouse, rat, and dog.

In some aspects, the present disclosure provides an isolated antibodythat specifically binds to an epitope similar to a ligand binding siteof human CTLA4. In some embodiments, the antibody specifically binds toan epitope similar to CD80 binding site of human CTLA4. In someembodiments, the antibody specifically binds to an epitope similar toCD86 binding site of human CTLA4. In some embodiments, the antibodyspecifically binds to an epitope comprising one or more amino acidresidues in a ligand binding site (e.g., CD80 and/or CD86 binding site)of human CTLA4. In some embodiments, the antibody specifically binds toan epitope on human CTLA4 that is different from the epitope ofipilimumab. In some embodiments, the epitope does not comprise aminoacid residues in the CC′ loop motif of human CTLA4. In some embodiments,the epitope does not comprise amino acid residue L106 or I108 of humanCTLA4. In some embodiments, the antibody specifically binds to anepitope comprising amino acid residues Y105 and L106, but not I108 ofhuman CTLA4, wherein the numbering of the amino acid residues isaccording to SEQ ID NO: 207.

(SEQ ID NO: 207) KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVEL MYPPPYYLGIGNGTQIYVIDPE

In one aspect, the present disclosure provides an isolated antibodycomprising a heavy chain variable region and a light chain variableregion, a) where the heavy chain variable region comprises an HVR-H1, anHVR-H2, and an HVR-H3, where the HVR-H1 comprises an amino acid sequenceaccording to a formula selected from: Formula (I): X1TFSX2YX3IHWV (SEQID NO: 1), where X1 is F or Y, X2 is D or G, and X3 is A, G, or W;Formula (II): YSIX1SGX2X3WX4WI (SEQ ID NO: 2), where X1 is S or T, X2 isH or Y, X3 is H or Y, and X4 is A, D, or S; and Formula (III):FSLSTGGVAVX1WI (SEQ ID NO: 3), where X1 is G or S; the HVR-H2 comprisesan amino acid sequence according to a formula selected from: Formula(IV): IGX1IX2HSGSTYYSX3SLKSRV (SEQ ID NO: 4), where X1 is D or E, X2 isS or Y, and X3 is P or Q; Formula (V): IGX1ISPSX2GX3TX4YAQKFQGRV (SEQ IDNO: 5), where X1 is I or W, X2 is G or S, X3 is G or S, and X4 is K orN; and Formula (VI): VSX1ISGX2GX3X4TYYADSVKGRF (SEQ ID NO: 6), where X1is A, G, or S, X2 is S or Y, X3 is G or S, and X4 is S or T; and theHVR-H3 comprises an amino acid sequence according to a formula selectedfrom: Formula (VII): ARX1X2X3X4FDX5 (SEQ ID NO: 7), where X1 is G, R, orS, X2 is A, I, or Y, X3 is D, V, or Y, X4 is A, E, or Y, and X5 is I orY; Formula (VIII): ARX1GX2GYFDX3 (SEQ ID NO: 8), where X1 is D or L, X2is F or Y, and X3 is V or Y; Formula (IX): ARX1X2X3X4AX5X6FDY (SEQ IDNO: 9), where X1 is L or R, X2 is I or P, X3 is A or Y, X4 is S or T, X5is T or Y, and X6 is A or Y; and Formula (X): ARDX1X2X3GSSGYYX4GFDX5(SEQ ID NO: 10), where X1 is I or V, X2 is A or H, X3 is P or S, X4 is Dor Y, and X5 is F or V; and/or b) where the light chain variable regioncomprises an HVR-L1, an HVR-L2, and an HVR-L3, where the HVR-L1comprises an amino acid sequence according to a formula selected from:Formula (XI): RASQX1X2X3SX4LX5 (SEQ ID NO: 11), where X1 is G or S, X2is I or V, X3 is G or S, X4 is S or Y, and X5 is A or N; Formula (XII):RASQX1VX2X3RX4LA (SEQ ID NO: 12), where X1 is S or T, X2 is F, R, or S,X3 is G or S, and X4 is F or Y; and Formula (XIII): RASX1SVDFX2GX3SFLX4(SEQ ID NO: 13), where X1 is E or Q, X2 is D, F, H, or Y, X3 is F, I, orK, and X4 is A, D, or H; the HVR-L2 comprises an amino acid sequenceaccording to Formula (XIV): X1ASX2X3X4X5GX6 (SEQ ID NO: 14), where X1 isA or D, X2 is N, S, or T, X3 is L or R, X4 is A, E, or Q, X5 is S or T,and X6 is I or V; and the HVR-L3 comprises an amino acid sequenceaccording to a formula selected from: Formula (XV): YCX1X2X3X4X5X6PX7T(SEQ ID NO: 15), where X1 is E, Q, or V, X2 is H or Q, X3 is A, G, H, R,or S, X4 is D, L, S, or Y, X5 is E, G, P, Q, or S, X6 is L, T, V, or W,and X7 is F, L, P, W, or Y; Formula (XVI): YCQQX1X2X3WPPWT (SEQ ID NO:16), where X1 is S or Y, X2 is D or Y, and X3 is Q or Y; and Formula(XVII): YCQX1YX2SSPPX3YT (SEQ ID NO: 17), where X1 is H or Q, X2 is T orV, and X3 is E or V.

In some embodiments, the antibody comprises: a) an HVR-H1 comprising anamino acid sequence selected from SEQ ID NOS: 18-29; an HVR-H2comprising an amino acid sequence selected from SEQ ID NOS: 30-39; andan HVR-H3 comprising an amino acid sequence selected from SEQ ID NOS:40-52; and/or b) an HVR-L1 comprising an amino acid sequence selectedfrom SEQ ID NOS: 53-65; an HVR-L2 comprising an amino acid sequenceselected from SEQ ID NOS: 66-69; and an HVR-L3 comprising an amino acidsequence selected from SEQ ID NOS: 70-81. In some embodiments, theantibody comprises one, two, three, four, five, or all six of the HVRsshown for any of the exemplary antibodies described in Table A below.

TABLE A anti-CTLA4 HVR sequences Ab name: HVR-H1 HVR-H2 HVR-H3 HVR-L1HVR-L2 HVR-L3 TY21 FTFSDYAIH IGIISPSSGSTNYAQ ARDIHSGSSGY RASESVDFFGDASNR YCQHYTSS 585 WV KFQGRV YYGFDV ISFLA ATGI PPVYT (SEQ ID NO:(SEQ ID NO: 30) (SEQ ID NO: 40) (SEQ ID NO: (SEQ ID (SEQ ID NO: 18) 53)NO: 66) 70) TY21 YSITSGYY VSSISGSGSTTYYA ARDGFGYFDY SASSSVSYVY DASSLEYCVQGLQT 586 WAWI DSVKGRF (SEQ ID NO: 41) (SEQ ID NO: SGV PWT(SEQ ID NO: (SEQ ID NO: 31) 54) (SEQ ID (SEQ ID NO: 19) NO: 67) 71) TY21FTFSDYGIH IGEIYHSGSTYYSP ARDVAPGSSGY RASQGIGSSL DASNR YCQQYDQ 587 WVSLKSRV YDGFDF A ATGI WPPWT (SEQ ID NO: (SEQ ID NO: 32) (SEQ ID NO: 42)(SEQ ID NO: (SEQ ID (SEQ ID NO: 20) 55) NO: 66) 72) TY21 YSISSGYHVSGISGYGGSTYY ARHSYYGSGNF RASESVDFFG DASNL YCQQSYS 588 WDWI ADSVKGRF DYKSFLH ETGV WPPT (SEQ ID NO: (SEQ ID NO: 33) (SEQ ID NO: 43) (SEQ ID NO:(SEQ ID (SEQ ID NO: 21) 56) NO: 68) 73) TY21 FTFSDYWI IGWISPSGGGTKYAARGAYEFDY RASQSVSSRF DASNR YCQQSYPT 589 HWV QKFQGRV (SEQ ID NO: 44) LAATGI PLT (SEQ ID NO: (SEQ ID NO: 34) (SEQ ID NO: (SEQ ID (SEQ ID NO: 22)57) NO: 66) 74) TY21 YSISSGYH LARIDWDDDKYYS ARSYVYFDY RASQSVRGR DASNRYCQQSSSW 580 WSWI TSLKSRL (SEQ ID NO: 45) FLA ATGI PPT (SEQ ID NO:(SEQ ID NO: 35) (SEQ ID NO: (SEQ ID (SEQ ID NO: 23) 58) NO: 66) 75) TY21FSLSTGGV IGEIYHSGSTYYSP ARRIATATYFD RASQTVFSR DASNR YCQQSYY 591 AVSWISLKSRV Y YLA ATGI WPPWT (SEQ ID NO: (SEQ ID NO: 32) (SEQ ID NO: 46)(SEQ ID NO: (SEQ ID (SEQ ID NO: 24) 59) NO: 66) 76) TY21 FSLSTGGVVSAISGYGSTTYY ARLPYSAYAFD RASQGVSSY AASTL YCQHHYG 686 AVGWI ADSVKGRF YLA QSGV TPLT (SEQ ID NO: (SEQ ID NO: 36) (SEQ ID NO: 47) (SEQ ID NO:(SEQ ID (SEQ ID NO: 25) 60) NO: 69) 77) TY21 FTFSGYAIH IGIISPSGGGTKYAARHPFAY RASQSVDFY DASNR YCQQYVSS 687 WV QKFQGRV (SEQ ID NO: 48) GISFLDATGI PPEYT (SEQ ID NO: (SEQ ID NO: 37) (SEQ ID NO: (SEQ ID (SEQ ID NO:26) 61) NO: 66) 78) TY21 YTFSGYGI IGEIYHSGSTYYSP ARRIDAFDI RASQSVDFDDASSLE YCQQRDS 689 HWV SLKSRV (SEQ ID NO: 49) GFSFLH SGV WPYT(SEQ ID NO: (SEQ ID NO: 32) (SEQ ID NO: (SEQ ID (SEQ ID NO: 27) 62)NO: 67) 79) TY21 YTFSGYAI IGIISPSGGGTKYA ARLYDVAY RASQSVDFH DASSLEYCEQSLEV 680 HWV QKFQGRV (SEQ ID NO: 50) GKSFLH SGV PFT (SEQ ID NO:(SEQ ID NO: 37) (SEQ ID NO: (SEQ ID (SEQ ID NO: 28) 63) NO: 67) 80) TY21FTFSDYAIH IGIISPSGGSTKYAQ ARLGYGYFDV RASQSVDFY DASSLE YCVQALQL 691 WVKFQGRV (SEQ ID NO: 51) GISFLH SGV PLT (SEQ ID NO: (SEQ ID NO: 38)(SEQ ID NO: (SEQ ID (SEQ ID NO: 18) 64) NO: 67) 81) TY21 YSITSGHYIGDISHSGSTYYSQ ARGSRTGYFDY RASQSISSYL DASNL YCQHHYG 692 WSWI SLKSRV(SEQ ID NO: 52) N ETGV TPLT (SEQ ID NO: (SEQ ID NO: 39) (SEQ ID NO:(SEQ ID (SEQ ID NO: 29) 65) NO: 68) 77)

In some embodiments, the antibody comprises an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 18, an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 30, an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 40, an HVR-L1 comprising the amino acid sequenceof SEQ ID NO: 53, an HVR-L2 comprising the amino acid sequence of SEQ IDNO: 66, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:70. In some embodiments, the antibody comprises an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 19, an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 31, an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 41, an HVR-L1 comprising the amino acid sequenceof SEQ ID NO: 54, an HVR-L2 comprising the amino acid sequence of SEQ IDNO: 67, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:71. In some embodiments, the antibody comprises an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 20, an HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 32, an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 42, an HVR-L1 comprising the amino acid sequenceof SEQ ID NO: 55, an HVR-L2 comprising the amino acid sequence of SEQ IDNO: 66, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:72. In some embodiments, the antibody comprises an HVR-H1 comprising theamino acid sequence of SEQ ID NO: 21 an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 33, an HVR-H3 comprising the amino acid sequenceof SEQ ID NO: 43, an HVR-L1 comprising the amino acid sequence of SEQ IDNO: 56, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 68,and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 73. Insome embodiments, the antibody comprises an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 22, an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 34, an HVR-H3 comprising the amino acid sequenceof SEQ ID NO: 44, an HVR-L1 comprising the amino acid sequence of SEQ IDNO: 57, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66,and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74. Insome embodiments, the antibody comprises an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 23, an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 35, an HVR-H3 comprising the amino acid sequenceof SEQ ID NO: 45, an HVR-L1 comprising the amino acid sequence of SEQ IDNO: 58, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66,and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 75. Insome embodiments, the antibody comprises an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 24, an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 32, an HVR-H3 comprising the amino acid sequenceof SEQ ID NO: 46, an HVR-L1 comprising the amino acid sequence of SEQ IDNO: 59, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66,and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76. Insome embodiments, the antibody comprises an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 25, an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 36, an HVR-H3 comprising the amino acid sequenceof SEQ ID NO: 47, an HVR-L1 comprising the amino acid sequence of SEQ IDNO: 60, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 69,and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 77. Insome embodiments, the antibody comprises an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 26, an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 37, an HVR-H3 comprising the amino acid sequenceof SEQ ID NO: 48, an HVR-L1 comprising the amino acid sequence of SEQ IDNO: 61, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66,and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 78. Insome embodiments, the antibody comprises an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 27, an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 32, an HVR-H3 comprising the amino acid sequenceof SEQ ID NO: 49, an HVR-L1 comprising the amino acid sequence of SEQ IDNO: 62, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 67,and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 79. Insome embodiments, the antibody comprises an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 28, an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 37, an HVR-H3 comprising the amino acid sequenceof SEQ ID NO: 50, an HVR-L1 comprising the amino acid sequence of SEQ IDNO: 63, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 67,and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 80. Insome embodiments, the antibody comprises an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 18, an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 38, an HVR-H3 comprising the amino acid sequenceof SEQ ID NO: 51, an HVR-L1 comprising the amino acid sequence of SEQ IDNO: 64, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 67,and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 81. Insome embodiments, the antibody comprises an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 29, an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 39, an HVR-H3 comprising the amino acid sequenceof SEQ ID NO: 52, an HVR-L1 comprising the amino acid sequence of SEQ IDNO: 65, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 68,and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 77.

In some embodiments, the antibody comprises: a) a heavy chain variableregion comprising an amino acid sequence selected from SEQ ID NOS:82-94; and/or b) a light chain variable region comprising an amino acidsequence selected from SEQ ID NOS: 95-107. In some embodiments, theantibody comprises a heavy chain variable region comprising an aminoacid sequence having at least 90% (e.g., at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to a sequenceselected from SEQ ID NOS: 82-94, and/or a light chain variable regioncomprising an amino acid sequence having at least 90% (e.g., at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identityto a sequence selected from SEQ ID NOS: 95-107. In some embodiments, theantibody comprises a heavy chain variable region and a light chainvariable region of any of the exemplary antibodies described in Table Bbelow. In some embodiments, the antibody comprises one, two, or allthree HVRs of the heavy chain variable region, and/or one, two, or allthree HVRs of the light chain variable region shown for any of theexemplary antibodies described in Table B below.

TABLE B anti-CTLA4 variable region amino acid sequences Ab name: VH: VL:TY21585 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIQLTQSPSSLSASVGDRVTITCRASESVDFF AIHWVRQAPGKGLEWIGIISPSSGSTNYAQKFGISFLAWYQQKPGKAPKLLIYDASNRATGI QGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ ARDIHSGSSGYYYGFDVWGQGTLVTVSSHYTSSPPVYTFGQGTKVEIKR (SEQ ID NO: 82) (SEQ ID NO: 95) TY21586EVQLVESGGGLVQPGGSLRLSCAASGYSITSG DIQLTQSPSSLSASVGDRVTITCSASSSVSYVYYWAWIRQAPGKGLEWVSSISGSGSTTYYAD YWYQQKPGKAPKLLIYDASSLESGVPSRFSSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVY GSGSGTDFTLTISSLQPEDFATYYCVQGLQTYCARDGFGYFDYWGQGTLVTVSS PWTFGQGTKVEIKR (SEQ ID NO: 83) (SEQ ID NO: 96)TY21587 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIQLTQSPSSLSASVGDRVTITCRASQGIGSS GIHWVRQAPGKGLEWIGEIYHSGSTYYSPSLKLAWYQQKPGKAPKLLIYDASNRATGIPSRF SRVTISRDNSKNTLYLQLNSLRAEDTAVYYCASGSGSGTDFTLTISSLQPEDFATYYCQQYDQ RDVAPGSSGYYDGFDFWGQGTLVTVSSWPPWTFGQGTKVEIKR (SEQ ID NO: 84) (SEQ ID NO: 97) TY21588EVQLVESGGGLVQPGGSLRLSCAASGYSISSG DIQLTQSPSSLSASVGDRVTITCRASESVDFFYHWDWIRQAPGKGLEWVSGISGYGGSTYYA GKSFLHWYQQKPGKAPKLLIYDASNLETGDSVKGRFTISRDNSKNTLYLQLNSLRAEDTAV VPSRFSGSGSGTDFTLTISSLQPEDFATYYCYYCARHSYYGSGNFDYWGQGTLVTVSS QQSYSWPPTFGQGTKVEIKR (SEQ ID NO: 85)(SEQ ID NO: 98) TY21589 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIQLTQSPSSLSASVGDRVTITCRASQSVSS WIHWVRQAPGKGLEWIGWISPSGGGTKYAQKRFLAWYQQKPGKAPKLLIYDASNRATGIPS FQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYRFSGSGSGTDFTLTISSLQPEDFATYYCQQS CARGAYEFDYWGQGTLVTVSS YPTPLTFGQGTKVEIKR(SEQ ID NO: 86) (SEQ ID NO: 99) TY21580 EVQLVESGGGLVQPGGSLRLSCAASGYSISSGDIQLTQSPSSLSASVGDRVTITCRASQSVRG YHWSWIRQAPGKGLEWLARIDWDDDKYYSTRFLAWYQQKPGKAPKLLIYDASNRATGIPS SLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYRFSGSGSGTDFTLTISSLQPEDFATYYCQQS CARSYVYFDYWGQGTLVTVSS SSWPPTFGQGTKVEIKR(SEQ ID NO: 87) (SEQ ID NO: 100) TY21591EVQLVESGGGLVQPGGSLRLSCAASGFSLSTG DIQLTQSPSSLSASVGDRVTITCRASQTVFSGVAVSWIRQAPGKGLEWIGEIYHSGSTYYSPS RYLAWYQQKPGKAPKLLIYDASNRATGIPSLKSRVTISRDNSKNTLYLQLNSLRAEDTAVYY RFSGSGSGTDFTLTISSLQPEDFATYYCQQSCARRIATATYFDYWGQGTLVTVSS YYWPPWTFGQGTKVEIKR (SEQ ID NO: 88)(SEQ ID NO: 101) TY21686 EVQLVESGGGLVQPGGSLRLSCAASGFSLSTGDIQLTQSPSSLSASVGDRVTITCRASQGVSS GVAVGWIRQAPGKGLEWVSAISGYGSTTYYAYLAWYQQKPGKAPKLLIYAASTLQSGVPSR DSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVFSGSGSGTDFTLTISSLQPEDFATYYCQHHY YYCARLPYSAYAFDYWGQGTLVTVSSGTPLTFGQGTKVEIKR (SEQ ID NO: 89) (SEQ ID NO: 102) TY21687EVQLVESGGGLVQPGGSLRLSCAASGFTFSGY DIQLTQSPSSLSASVGDRVTITCRASQSVDFAIHWVRQAPGKGLEWIGIISPSGGGTKYAQKF YGISFLDWYQQKPGKAPKLLIYDASNRATGQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYC IPSRFSGSGSGTDFTLTISSLQPEDFATYYCQARHPFAYWGQGTLVTVSS QYVSSPPEYTFGQGTKVEIKR (SEQ ID NO: 90)(SEQ ID NO: 103) TY21689 EVQLVESGGGLVQPGGSLRLSCAASGYTFSGYDIQLTQSPSSLSASVGDRVTITCRASQSVDF GIHWVRQAPGKGLEWIGEIYHSGSTYYSPSLKDGFSFLHWYQQKPGKAPKLLIYDASSLESG SRVTISRDNSKNTLYLQLNSLRAEDTAVYYCAVPSRFSGSGSGTDFTLTISSLQPEDFATYYC RRIDAFDIWGQGTLVTVSS QQRDSWPYTFGQGTKVEIKR(SEQ ID NO: 91) (SEQ ID NO: 104) TY21680EVQLVESGGGLVQPGGSLRLSCAASGYTFSGY DIQLTQSPSSLSASVGDRVTITCRASQSVDFAIHWVRQAPGKGLEWIGIISPSGGGTKYAQKF HGKSFLHWYQQKPGKAPKLLIYDASSLESQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYC GVPSRFSGSGSGTDFTLTISSLQPEDFATYYARLYDVAYVVGQGTLVTVSS CEQSLEVPFTFGQGTKVEIKR (SEQ ID NO: 92)(SEQ ID NO: 105) TY21691 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYDIQLTQSPSSLSASVGDRVTITCRASQSVDF AIHWVRQAPGKGLEWIGIISPSGGSTKYAQKFYGISFLHWYQQKPGKAPKLLIYDASSLESG QGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCVPSRFSGSGSGTDFTLTISSLQPEDFATYYC ARLGYGYFDVVVGQGTLVTVSSVQALQLPLTFGQGTKVEIKR (SEQ ID NO: 93) (SEQ ID NO: 106) TY21692EVQLVESGGGLVQPGGSLRLSCAASGYSITSG DIQLTQSPSSLSASVGDRVTITCRASQSISSYHYWSWIRQAPGKGLEWIGDISHSGSTYYSQSL LNWYQQKPGKAPKLLIYDASNLETGVPSRKSRVTISRDNSKNTLYLQLNSLRAEDTAVYYC FSGSGSGTDFTLTISSLQPEDFATYYCQHHYARGSRTGYFDYWGQGTLVTVSS GTPLTFGQGTKVEIKR (SEQ ID NO: 94) (SEQ ID NO: 107)

In some embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 82, and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:95. In some embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 83, and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:96. In some embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 84, and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:97. In some embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 85, and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:98. In some embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 86, and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:99. In some embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 87, and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:100. In some embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 88, and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:101. In some embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 89, and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:102. In some embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 90, and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:103. In some embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 91, and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:104. In some embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 92, and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:105. In some embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 93, and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:106. In some embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 94, and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:107.

In some embodiments, an antibody of the present disclosurecross-competes for binding to human CTLA4 with an antibody comprising:a) an HVR-H1 comprising an amino acid sequence selected from SEQ ID NOS:18-29; an HVR-H2 comprising an amino acid sequence selected from SEQ IDNOS: 30-39; and an HVR-H3 comprising an amino acid sequence selectedfrom SEQ ID NOS: 40-52; and/or b) an HVR-L1 comprising an amino acidsequence selected from SEQ ID NOS: 53-65; an HVR-L2 comprising an aminoacid sequence selected from SEQ ID NOS: 66-69; and an HVR-L3 comprisingan amino acid sequence selected from SEQ ID NOS: 70-81. In someembodiments, an antibody of the present disclosure cross-competes forbinding to human CTLA4 with an antibody comprising one, two, three,four, five, or all six of the HVRs shown for any of the exemplaryantibodies described in Table A. In some embodiments, an antibody of thepresent disclosure cross-competes for binding to human CTLA4 with anantibody comprising: a) a heavy chain variable region comprising anamino acid sequence selected from SEQ ID NOS: 82-94; and/or b) a lightchain variable region comprising an amino acid sequence selected fromSEQ ID NOS: 95-107. In some embodiments, an antibody of the presentdisclosure cross-competes for binding to human CTLA4 with an antibodycomprising a VH and/or VL shown for any of the exemplary antibodiesdescribed in Table B.

The CTLA4 antibodies described herein may be in any class, such as IgG,IgM, IgE, IgA, or IgD. In some embodiments, the CTLA4 antibodies are inthe IgG class, such as IgG1, IgG2, IgG3, or IgG4 subclass. A CTLA4antibody can be converted from one class or subclass to another class orsubclass using methods known in the art. An exemplary method forproducing an antibody in a desired class or subclass comprises the stepsof isolating a nucleic acid encoding a heavy chain of a CTLA4 antibodyand a nucleic acid encoding a light chain of a CTLA4 antibody, isolatingthe sequence encoding the V_(H) region, ligating the V_(H) sequence to asequence encoding a heavy chain constant region of the desired class orsubclass, expressing the light chain gene and the heavy chain constructin a cell, and collecting the CTLA4 antibody. Antibodies of the presentdisclosure may be monoclonal antibodies or polyclonal antibodies.Antibodies of the present disclosure may be monospecific antibodies ormultispecific (e.g., bispecific, trispecific, etc.) antibodies. In someembodiments, the CTLA4 antibodies described herein may include one ormore Fc mutations (e.g., that modulate (increase or decrease) ADCC orCDC activities). Any suitable Fc mutations known in the art may be usedin the CTLA4 antibodies of the present disclosure.

In some embodiments, an antibody of the present disclosure is abispecific antibody that binds to a first and second target, where thefirst target is human CTLA4. In some embodiments, the bispecificantibody binds to a first and second target, where the first target ishuman CTLA4, and where the bispecific antibody comprises a) an HVR-H1comprising an amino acid sequence selected from SEQ ID NOS: 18-29; anHVR-H2 comprising an amino acid sequence selected from SEQ ID NOS:30-39; and an HVR-H3 comprising an amino acid sequence selected from SEQID NOS: 40-52; and/or b) an HVR-L1 comprising an amino acid sequenceselected from SEQ ID NOS: 53-65; an HVR-L2 comprising an amino acidsequence selected from SEQ ID NOS: 66-69; and an HVR-L3 comprising anamino acid sequence selected from SEQ ID NOS: 70-81. In someembodiments, the bispecific antibody binds to a first and second target,where the first target is human CTLA4, and where the bispecific antibodycomprises one, two, three, four, five, or all six of the HVRs shown forany of the exemplary antibodies described in Table A. In someembodiments, the bispecific antibody binds to a first and second target,where the first target is human CTLA4, and where the bispecific antibodycomprises: a) a heavy chain variable region comprising an amino acidsequence selected from SEQ ID NOS: 82-94; and/or b) a light chainvariable region comprising an amino acid sequence selected from SEQ IDNOS: 95-107. In some embodiments, the bispecific antibody binds to afirst and second target, where the first target is human CTLA4, andwhere the bispecific antibody comprises a VH and/or VL shown for any ofthe exemplary antibodies described in Table B. In some embodiments, thesecond target is PD-1, PD-L1, PD-L2, LAG3, TIM3, B7-H3, CD95, CD120a,OX40, CD40, BTLA, VISTA, ICOS, Her1, Her2, Her3, or B7-H4.

Antibodies of the present disclosure may be produced by any techniquesknown in the art, including conventional monoclonal antibody methodologye.g., a standard somatic cell hybridization technique (see e.g., Kohlerand Milstein, Nature 256:495 (1975)), viral or oncogenic transformationof B lymphocytes, or recombinant antibody technologies as described indetail herein (see e.g., Examples 1 and 2). In some embodiments,antibodies of the present disclosure are produced using any of thelibraries and/or methods described in PCT application numberPCT/CN2017/098333 (incorporated herein by reference in its entirety)and/or PCT application number PCT/CN2017/098299 (incorporated herein byreference in its entirety).

Hybridoma production is a very well-established procedure. The commonanimal system for preparing hybridomas is the murine system.Immunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known. Onewell-known method that may be used for making human CTLA4 antibodiesprovided by the present disclosure involves the use of a XenoMouse™animal system. XenoMouse™ mice are engineered mouse strains thatcomprise large fragments of human immunoglobulin heavy chain and lightchain loci and are deficient in mouse antibody production (see e.g.,Green et al., (1994) Nature Genetics 7:13-21; WO2003/040170). The animalis immunized with a CTLA4 antigen. The CTLA4 antigen is isolated and/orpurified CTLA4. It may be a fragment of CTLA4, such as the extracellulardomain of CTLA4. Immunization of animals can be carried out by anymethod known in the art (see e.g., Harlow and Lane, Antibodies: ALaboratory Manual, New York: Cold Spring Harbor Press, 1990). Methodsfor immunizing non-human animals such as mice, rats, sheep, goats, pigs,cattle and horses are well known in the art (see e.g., Harlow and Lane,supra, and U.S. Pat. No. 5,994,619). The CTLA4 antigen may beadministered with an adjuvant to stimulate the immune response.Exemplary adjuvants include complete or incomplete Freund's adjuvant,RIBI (muramyl dipeptides) or ISCOM (immunostimulating complexes). Afterimmunization of an animal with a CTLA4 antigen, antibody-producingimmortalized cell lines are prepared from cells isolated from theimmunized animal. After immunization, the animal is sacrificed and lymphnode and/or splenic B cells are immortalized. Methods of immortalizingcells include, but are not limited to, transferring them with oncogenes,inflecting them with the oncogenic virus cultivating them underconditions that select for immortalized cells, subjecting them tocarcinogenic or mutating compounds, fusing them with an immortalizedcell, e.g., a myeloma cell, and inactivating a tumor suppressor gene(see e.g., Harlow and Lane, supra). If fusion with myeloma cells isused, the myeloma cells preferably do not secrete immunoglobulinpolypeptides (a non-secretory cell line). Immortalized cells arescreened using CTLA4, a portion thereof, or a cell expressing CTLA4.CTLA4 antibody-producing cells, e.g., hybridomas, are selected, clonedand further screened for desirable characteristics, including robustgrowth, high antibody production and desirable antibody characteristics,as discussed further below. Hybridomas can be expanded in vivo insyngeneic animals, in animals that lack an immune system, e.g., nudemice, or in cell culture in vitro. Methods of selecting, cloning andexpanding hybridomas are well known to those of ordinary skill in theart.

Antibodies of the present disclosure may also be prepared using phagedisplay or yeast display methods. Such display methods for isolatinghuman antibodies are established in the art (see e.g., Knappik, et al.(2000) J. Mol. Biol. 296, 57-86; Feldhaus et al. (2003) Nat Biotechnol21:163-170; see also the methods of Examples 1 and 2 below).

Antigen Binding Fragments

In some other aspects, the present disclosure provides antigen-bindingfragments of any of the CTLA4 antibodies described herein.

The antigen-binding fragment may comprise any sequences of any of theantibodies described herein. In some embodiments, the antigen-bindingfragment comprises the amino acid sequence of: (1) a light chain of aCTLA4 antibody; (2) a heavy chain of a CTLA4 antibody; (3) a variableregion from the light chain of a CTLA4 antibody; (4) a variable regionfrom the heavy chain of a CTLA4 antibody; (5) one or more HVRs (e.g.,one, two, three, four, five, or six HVRs) of a CTLA4 antibody; or (6)three HVRs from the light chain and three HVRs from the heavy chain of aCTLA4 antibody.

In some embodiments, the present disclosure provides an antigen-bindingfragment of an antibody selected from those listed in Tables A and B.

In some embodiments, the antigen-binding fragments of a CTLA4 antibodyinclude: (i) a Fab fragment, which is a monovalent fragment consistingof the V_(L), V_(H), C_(L) and C_(H)1 domains; (ii) a F(ab′)₂ fragment,which is a bivalent fragment comprising two Fab fragments linked by adisulfide bridge at the hinge region; (iii) a Fd fragment consisting ofthe V_(H) and C_(H)1 domains; (iv) a Fv fragment consisting of the V_(L)and V_(H) domains of a single arm of an antibody; (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a V_(H)domain; (vi) an isolated CDR, and (vii) single chain antibody (scFv),which is a polypeptide comprising a V_(L) region of an antibody linkedto a V_(H) region of an antibody (see e.g., Bird et al. (1988) Science242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883).

Antibody Derivatives

In some further aspects, the present disclosure provides derivatives ofany of the CTLA4 antibodies described herein.

In some embodiments, the antibody derivative is derived frommodifications of the amino acid sequences of an illustrative antibody(e.g., a “parent antibody”) of the present disclosure while conservingthe overall molecular structure of the parent antibody amino acidsequence. Amino acid sequences of any regions of the parent antibodychains may be modified, such as framework regions, HVR regions, orconstant regions. Types of modifications include substitutions,insertions, deletions, or combinations thereof, of one or more aminoacids of the parent antibody.

In some embodiments, the antibody derivative comprises a V_(L) or V_(H)region that is at least 65%, at least 75%, at least 85%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to anamino acid sequence as set forth in any of SEQ ID NOS: 82-107 In someembodiments, the antibody derivative comprises an HVR-H1 amino acidsequence region that is at least 65%, at least 75%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to an amino acid sequence as set forth in any of SEQ ID NOS:18-29. In some embodiments, the antibody derivative comprises an HVR-H2amino acid sequence region that is at least 65%, at least 75%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identical to an amino acid sequence as set forth in any of SEQ IDNOS: 30-39. In some embodiments, the antibody derivative comprises anHVR-H3 amino acid sequence region that is at least 65%, at least 75%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical to an amino acid sequence as set forth in any of SEQID NOS: 40-52. In some embodiments, the antibody derivative comprises anHVR-L1 amino acid sequence region that is at least 65%, at least 75%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical to an amino acid sequence as set forth in any of SEQID NOS: 53-65. In some embodiments, the antibody derivative comprises anHVR-L2 amino acid sequence region that is at least 65%, at least 75%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical to an amino acid sequence as set forth in any of SEQID NOS: 66-69. In some embodiments, the antibody derivative comprises anHVR-L3 amino acid sequence region that is at least 65%, at least 75%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical to an amino acid sequence as set forth in any of SEQID NOS: 70-81.

In some particular embodiments, the derivative comprises 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative or non-conservativesubstitutions, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or15 additions and/or deletions to an amino acid sequence as set forth inany of SEQ ID NOS: 18-107.

Amino acid substitutions encompass both conservative substitutions andnon-conservative substitutions. The term “conservative amino acidsubstitution” means a replacement of one amino acid with another aminoacid where the two amino acids have similarity in certainphysico-chemical properties such as polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues involved. For example, substitutions typically may be madewithin each of the following groups: (a) nonpolar (hydrophobic) aminoacids, such as alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan, and methionine; (b) polar neutral aminoacids, such as glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; (c) positively charged (basic) amino acids,such as arginine, lysine, and histidine; and (d) negatively charged(acidic) amino acids, such as aspartic acid and glutamic acid.

The modifications may be made in any positions of the amino acidsequences of the antibody, including the HVRs, framework regions, orconstant regions. In one embodiment, the present disclosure provides anantibody derivative that contains the V_(H) and V_(L) HVR sequences ofan illustrative antibody of this disclosure, yet contains frameworksequences different from those of the illustrative antibody. Suchframework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the Genbank database or in the “VBase”human germline sequence database (Kaba et al., Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242 (1991); Tomlinson et al., J.Mol. Biol. 227:776-798 (1992); and Cox et al., Eur. J. Immunol.24:827-836 (1994)). Framework sequences that may be used in constructingan antibody derivative include those that are structurally similar tothe framework sequences used by illustrative antibodies of thedisclosure For example, the HVR-H1, HVR-H2, and HVR-H3 sequences, andthe HVR-L1, HVR-L2, and HVR-L3 sequences of an illustrative antibody canbe grafted onto framework regions that have the identical sequence asthat found in the germline immunoglobulin gene from which the frameworksequence derive, or the HVR sequences can be grafted onto frameworkregions that contain one or more mutations as compared to the germlinesequences.

In some embodiments, the antibody derivative is a chimeric antibodywhich comprises an amino acid sequence of an illustrative antibody ofthe disclosure. In one example, one or more HVRs from one or moreillustrative antibodies are combined with HVRs from an antibody from anon-human animal, such as mouse or rat. In another example, all of theHVRs of the chimeric antibody are derived from one or more illustrativeantibodies. In some particular embodiments, the chimeric antibodycomprises one, two, or three HVRs from the heavy chain variable regionand/or one, two, or three HVRs from the light chain variable region ofan illustrative antibody. Chimeric antibodies can be generated usingconventional methods known in the art.

Another type of modification is to mutate amino acid residues within theHVR regions of the V_(H) and/or V_(L) chain. Site-directed mutagenesisor PCR-mediated mutagenesis can be performed to introduce themutation(s) and the effect on antibody binding, or other functionalproperty of interest, can be evaluated in in vitro or in vivo assaysknown in the art. Typically, conservative substitutions are introduced.The mutations may be amino acid additions and/or deletions. Moreover,typically no more than one, two, three, four or five residues within anHVR region are altered. In some embodiments, the antibody derivativecomprises 1, 2, 3, or 4 amino acid substitutions in the heavy chain HVRsand/or in the light chain HVRs. In another embodiment, the amino acidsubstitution is to change one or more cysteines in an antibody toanother residue, such as, without limitation, alanine or serine. Thecysteine may be a canonical or non-canonical cysteine. In oneembodiment, the antibody derivative has 1, 2, 3, or 4 conservative aminoacid substitutions in the heavy chain HVR regions relative to the aminoacid sequences of an illustrative antibody.

Modifications may also be made to the framework residues within theV_(H) and/or V_(L) regions. Typically, such framework variants are madeto decrease the immunogenicity of the antibody. One approach is to “backmutate” one or more framework residues to the corresponding germlinesequence. An antibody that has undergone somatic mutation may containframework residues that differ from the germline sequence from which theantibody is derived. Such residues can be identified by comparing theantibody framework sequences to the germline sequences from which theantibody is derived. To return the framework region sequences to theirgermline configuration, the somatic mutations can be “back mutated” tothe germline sequence by, for example, site-directed mutagenesis orPCR-mediated mutagenesis.

In addition, modifications may also be made within the Fc region of anillustrative antibody, typically to alter one or more functionalproperties of the antibody, such as serum half-life, complementfixation, Fc receptor binding, and/or antigen-dependent cellularcytotoxicity. In one example, the hinge region of CH1 is modified suchthat the number of cysteine residues in the hinge region is altered,e.g., increased or decreased. This approach is described further in U.S.Pat. No. 5,677,425. The number of cysteine residues in the hinge regionof CH1 is altered to, for example, facilitate assembly of the light andheavy chains or to increase or decrease the stability of the antibody.In another case, the Fc hinge region of an antibody is mutated todecrease the biological half-life of the antibody.

Furthermore, an antibody of the present disclosure may be modified toalter its potential glycosylation site or pattern in accordance withroutine experimentation known in the art. In another aspect, the presentdisclosure provides a derivative of a CTLA4 antibody that contains atleast one mutation in a variable region of a light chain or heavy chainthat changes the pattern of glycosylation in the variable region. Suchan antibody derivative may have an increased affinity and/or a modifiedspecificity for binding an antigen. The mutations may add a novelglycosylation site in the V region, change the location of one or more Vregion glycosylation site(s), or remove a pre-existing V regionglycosylation site. In one embodiment, the present disclosure provides aderivative of a CTLA4 antibody having a potential N-linked glycosylationsite at asparagine in the heavy chain variable region, wherein thepotential N-linked glycosylation site in one heavy chain variable regionis removed. In another embodiment, the present disclosure provides aderivative of a CTLA4 antibody having a potential N-linked glycosylationsite at asparagine in the heavy chain variable region, wherein thepotential N-linked glycosylation site in both heavy chain variableregions is removed. Method of altering the glycosylation pattern of anantibody is known in the art, such as those described in U.S. Pat. No.6,933,368, the disclosure of which incorporated herein by reference.

In another aspect, the present disclosure provides an antibodyderivative that comprises a CTLA4 antibody, or antigen-binding fragmentthereof, as described herein, linked to an additional molecular entity.Examples of additional molecular entities include pharmaceutical agents,peptides or proteins, detection agent or labels, and antibodies.

In some embodiments, the antibody derivative comprises an antibody ofthe disclosure linked to a pharmaceutical agent. Examples ofpharmaceutical agents include cytotoxic agents or other cancertherapeutic agents, and radioactive isotopes. Specific examples ofcytotoxic agents include taxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents alsoinclude, for example, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). Examples of radioactive isotopesthat can be conjugated to antibodies for use diagnostically ortherapeutically include, but are not limited to, iodine¹³¹, indium¹¹¹,yttrium⁹⁰ and lutetium¹⁷⁷. Methods for linking an antibody to apharmaceutical agent are known in the art, such as using various linkertechnologies. Examples of linker types include hydrazones, thioethers,esters, disulfides and peptide-containing linkers. For furtherdiscussion of linkers and methods for linking therapeutic agents toantibodies see e.g., Saito et al., Adv. Drug Deliv. Rev. 55:199-215(2003); Trail, et al., Cancer Immunol. Immunother. 52:328-337 (2003);Payne, Cancer Cell 3:207-212 (2003); Allen, Nat. Rev. Cancer 2:750-763(2002); Pastan and Kreitman, Curr. Opin. Investig. Drugs 3:1089-1091(2002); Senter and Springer (2001) Adv. Drug Deliv. Rev. 53:247-264.

In some embodiments, the antibody derivative is a CTLA4 antibodymultimer, which is a multimeric form of a CTLA4 antibody, such asantibody dimers, trimers, or higher-order oligomers of monomericantibodies. Individual monomers within an antibody multimer may beidentical or different. In addition, individual antibodies within amultimer may have the same or different binding specificities.Multimerization of antibodies may be accomplished through naturalaggregation of antibodies. For example, some percentage of purifiedantibody preparations (e.g., purified IgG4 molecules) spontaneously formprotein aggregates containing antibody homodimers, and otherhigher-order antibody multimers. Alternatively, antibody homodimers maybe formed through chemical linkage techniques known in the art, such asthrough using crosslinking agents. Suitable crosslinkers include thosethat are heterobifunctional, having two distinctly reactive groupsseparated by an appropriate spacer (such asm-maleimidobenzoyl-N-hydroxysuccinimide ester, succinimidyl4-(maleimidomethyl)cyclohexane-1-carboxylate, and N-succinimidylS-acethylthio-acetate) or homobifunctional (such as disuccinimidylsuberate). Such linkers are commercially available from, for example,Pierce Chemical Company, Rockford, Ill. Antibodies can also be made tomultimerize through recombinant DNA techniques known in the art.

Examples of other antibody derivatives provided by the presentdisclosure include single chain antibodies, diabodies, domainantibodies, nanobodies, and unibodies. A “single-chain antibody” (scFv)consists of a single polypeptide chain comprising a V_(L) domain linkedto a V_(H) domain wherein V_(L) domain and V_(H) domain are paired toform a monovalent molecule. Single chain antibody can be preparedaccording to method known in the art (see e.g., Bird et al., (1988)Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). A “diabody” consists of two chains, each chain comprisinga heavy chain variable region connected to a light chain variable regionon the same polypeptide chain connected by a short peptide linker,wherein the two regions on the same chain do not pair with each otherbut with complementary domains on the other chain to form a bispecificmolecule. Methods of preparing diabodies are known in the art (see e.g.,Holliger P. et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448, andPoljak R. J. et al., (1994) Structure 2:1121-1123). Domain antibodies(dAbs) are small functional binding units of antibodies, correspondingto the variable regions of either the heavy or light chains ofantibodies. Domain antibodies are well expressed in bacterial, yeast,and mammalian cell systems. Further details of domain antibodies andmethods of production thereof are known in the art (see e.g., U.S. Pat.Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197; 6,696,245; EuropeanPatents 0368684 & 0616640; WO05/035572, WO04/101790, WO04/081026,WO04/058821, WO04/003019 and WO03/002609). Nanobodies are derived fromthe heavy chains of an antibody. A nanobody typically comprises a singlevariable domain and two constant domains (CH2 and CH3) and retainsantigen-binding capacity of the original antibody. Nanobodies can beprepared by methods known in the art (see e.g., U.S. Pat. Nos.6,765,087, 6,838,254, WO 06/079372). Unibodies consist of one lightchain and one heavy chain of an IgG4 antibody. Unibodies may be made bythe removal of the hinge region of IgG4 antibodies. Further details ofunibodies and methods of preparing them may be found in WO2007/059782.

IV. Activatable Binding Polypeptides Targeting CTLA4

The present disclosure also relates, in part, toprecision/context-dependent activatable binding polypeptides (i.e.,activatable antibodies) that bind to human CTLA4, including activatableantibodies comprising any of the anti-CTLA4 antibodies described herein(e.g., anti-CTLA4 antibodies, anti-CTLA4 antibody binding fragments,and/or anti-CTLA4 antibody derivatives), antigen binding fragments ofthe activatable anti-CTLA4 antibodies, and/or derivatives of theactivatable anti-CTLA4 antibodies. In some embodiments, the activatableanti-CTLA4 antibodies described herein may have improved safetyprofiles. For example, the anti-CTLA4 antibodies described herein mayhave better safety margin as assessed by spleen weight change. Thechange in spleen size with the increase in drug dose administered isused as a benchmark to assess the safety margin of the drug candidateused. As shown in FIG. 48A-B, the activatable anti-CTLA4 antibodiesdescribed herein have a better safety margin relative to the parentalantibody (the antibody without the masking moiety).

In some embodiments, an activatable antibody of the present disclosurecomprises: (a) a masking moiety (MM); (b) a cleavable moiety (CM); and(c) a target binding moiety (TBM). In some embodiments, the MM is any ofthe masking moieties described herein. In some embodiments, the CM isany of the cleavable moieties described herein. In some embodiments, theTBM is any of the target binding moieties described herein (e.g., atarget binding moiety (TBM) comprising an antibody light chain variableregion and/or an antibody heavy chain variable region, such as a VHand/or VL of any of the anti-CTLA4 antibodies described herein). In someembodiments, the MM interferes with and/or inhibits the binding of theactivatable antibody to its target (e.g., human CTLA4 or human CD137)when the CM is not cleaved. In some embodiments, the activatableantibody is capable of binding to its target (e.g., human CTLA4 or humanCD137) when the CM is cleaved.

In some embodiments, the activatable antibody comprises: (a) apolypeptide comprising, from N-terminus to C-terminus, a masking moiety(MM), a cleavable moiety (CM), and a target binding moiety (TBM), wherethe MM is any of the masking moieties described herein, the CM is any ofthe cleavable moieties described herein, and where the TBM comprises anantibody light chain variable region (VL); and (b) an antibody heavychain variable region (VH).

In some embodiments, the activatable antibody comprises: (a) apolypeptide comprising, from N-terminus to C-terminus, a masking moiety(MM), a cleavable moiety (CM), and a target binding moiety (TBM), wherethe MM is any of the masking moieties described herein, the CM is any ofthe cleavable moieties described herein, and where the TBM comprises anantibody heavy chain variable region (VH); and (b) an antibody lightchain variable region (VL).

In some embodiments, the activatable antibody comprises: a polypeptidecomprising, from N-terminus to C-terminus, a masking moiety (MM), acleavable moiety (CM), and a target binding moiety (TBM), where the MMis any of the masking moieties described herein, the CM is any of thecleavable moieties described herein, and where the TBM comprises anantibody heavy chain variable region (VH) and an antibody light chainvariable region (VL).

The term “activatable binding polypeptide”, “ABP”, or “activatableantibody” includes a polypeptide that comprises a target binding moiety(TBM), a cleavable moiety (CM), and a masking moiety (MM). In someembodiments, the TBM comprises an amino acid sequence that binds to atarget. In some embodiments, the TBM comprises an antigen binding domain(ABD) of an antibody or antibody fragment thereof (e.g., any of theantibodies or antigen binding fragments described herein). In someembodiments, the antigen binding domain comprises a heavy chain variableregion comprising one, two, or three of the heavy chain variable regionHVRs described herein, and a light chain variable region comprising one,two, or three of the light chain variable region HVRs described herein(e.g., one, two, or three of the heavy chain variable region HVRsequences, and/or one, two, or three of the light chain variable regionHVR sequences as shown in Table A, including all six HVRs of any of theexemplary antibodies as shown in Table A). In some embodiments, theantigen binding domain comprises a heavy chain variable regioncomprising any of the heavy chain variable region sequences describedherein, and a light chain variable region comprising any of the lightchain variable region sequences described herein (e.g., a heavy chainvariable region sequence and/or a light chain variable region sequenceas shown in Table B). In some embodiments, the TBM (e.g., comprising anABD) comprises an antibody light chain variable region (VL) and anantibody heavy chain variable region (VH), wherein the VH and VL forms abinding domain that binds to the target in the absence of the MM. Insome embodiments, the VH and VL are covalently linked, e.g., in an scFv.In some embodiments, the VH and VL are not covalently linked. In someembodiments, the VH and VL form a Fab fragment. In some embodiments, theVH is linked to an antibody heavy chain constant region, and the VL islinked to an antibody light chain constant region.

In some embodiments, the activatable antibody comprises a polypeptidecomprising the structure, from N-terminus to C-terminus, of: maskingmoiety (MM)-cleavable moiety (CM)-VL, and the activatable antibodyfurther comprises a second polypeptide comprising a VH (e.g., a Fabfragment). In some embodiments, the activatable antibody comprises apolypeptide comprising the structure, from N-terminus to C-terminus, of:masking moiety (MM)-cleavable moiety (CM)-VL-VH (e.g., an scFv). In someembodiments, the activatable antibody comprises a polypeptide comprisingthe structure, from N-terminus to C-terminus, of: masking moiety(MM)-cleavable moiety (CM)-VH, and the activatable antibody furthercomprises a second polypeptide comprising a VL (e.g., a Fab fragment).In some embodiments, the activatable antibody comprises a polypeptidecomprising the structure, from N-terminus to C-terminus, of: maskingmoiety (MM)-cleavable moiety (CM)-VH-VL (e.g., an scFv).

The CM generally includes an amino acid sequence that is cleavable, forexample, serves as the substrate for an enzyme and/or acysteine-cysteine pair capable of forming a reducible disulfide bond. Assuch, when the terms “cleavage,” “cleavable,” “cleaved” and the like areused in connection with a CM, the terms encompass enzymatic cleavage,e.g., by a protease, as well as disruption of a disulfide bond between acysteine-cysteine pair via reduction of the disulfide bond that canresult from exposure to a reducing agent.

The MM refers to an amino acid sequence that, when the CM of theactivatable antibody is intact (e.g., uncleaved by a correspondingenzyme, and/or containing an unreduced cysteine-cysteine disulfidebond), the MM interferes with or inhibits binding of the TBM to itstarget. In some embodiments, the MM interferes with or inhibits bindingof the TBM to its target so efficiently that binding of the TBM to itstarget is extremely low and/or below the limit of detection (e.g.,binding cannot be detected in an ELISA or flow cytometry assay). Theamino acid sequence of the CM may overlap with or be included within theMM. It should be noted that for sake of convenience “ABP” or“activatable antibody” are used herein to refer to an ABP or activatableantibody in both their uncleaved (or “native”) state, as well as intheir cleaved state. It will be apparent to the ordinarily skilledartisan that in some embodiments a cleaved ABP may lack an MM due tocleavage of the CM, e.g., by a protease, resulting in release of atleast the MM (e.g., where the MM is not joined to the ABP by a covalentbond (e.g., a disulfide bond between cysteine residues)). Exemplary ABPsare described in more detail below.

In some embodiments, the masking moiety (MM) interferes with, obstructs,reduces the ability of, prevents, inhibits, or competes with the targetbinding moiety for binding to its target (e.g., an “inactive activatableantibody). In some embodiments, the masking moiety (MM) interferes with,obstructs, reduces, prevents, inhibits, or competes with the targetbinding moiety for binding to its target only when the polypeptide hasnot been activated (e.g., activated by a change in pH (increased ordecreased), activated by a temperature shift (increased or decreased),activated after being contacted with a second molecule (such as a smallmolecule or a protein ligand), etc.). In some embodiments, activationinduces cleavage of the polypeptide within the cleavage moiety. In someembodiments, activation induces conformation changes in the polypeptide(e.g., displacement of the masking moiety (MM)), leading to the maskingmoiety no longer preventing the activatable antibody from binding to itstarget. In some embodiments, the masking moiety (MM) interferes with,obstructs, reduces the ability of, prevents, inhibits, or competes withthe target binding moiety for binding to its target only when thecleavable moiety (CM) has not been cleaved by one or more proteases thatcleave within the cleavable moiety (CM). In some embodiments, themasking moiety (MM) has a masking efficiency of at least about 2.0(e.g., at least about 2.0, at least about 3.0, at least about 4.0, atleast about 5.0, at least about 6.0, at least about 7.0, at least about8.0, at least about 9.0, at least about 10, at least about 25, at leastabout 50, at least about 75, at least about 100, at least about 150, atleast about 200, at least about 300, at least about 400, at least about500, etc.) prior to activation. In some embodiments, masking efficiencyis measured as the difference in affinity of an activatable antibodycomprising the masking moiety (MM) for binding its target (beforeactivation) relative to the affinity of a polypeptide lacking themasking moiety for binding its target (e.g., the difference in affinityfor a target antigen (such as CTLA4) of an activatable antibodycomprising a masking moiety (MM) (before activation) relative to aparental antibody lacking the masking moiety (MM), or the difference inaffinity for a target antigen (such as CTLA4) of an activatable antibodycomprising a masking moiety (MM) (before activation) relative to theaffinity for the target antigen of the activatable antibody afteractivation). In some embodiments, the masking efficiency is measured bydividing the EC₅₀ for binding of an activatable antibody comprising amasking moiety (MM) (before activation) by the EC₅₀ of the parentalantibody (e.g., by measuring EC₅₀ by ELISA; see e.g., the methods ofExample 8). In some embodiments, masking efficiency is measured as thedifference in affinity of an activatable antibody comprising the maskingmoiety (MM) for binding its target before activation relative to theaffinity of the activatable antibody comprising the masking moiety (MM)for binding its target after activation (e.g., the difference inaffinity for a target antigen (such as CTLA4) of an activatable antibodybefore activation relative to the activatable antibody afteractivation). In some embodiments, the masking moiety (MM) binds to thetarget binding moiety (TBM), and prevents the activatable antibody frombinding to its target (e.g., an “inactive” activatable antibody). Insome embodiments, the masking moiety (MM) has a dissociation constantfor binding to the target binding moiety (TBM) that is greater than thedissociation constant of the target binding moiety (TBM) for its target.

In some embodiments, the masking moiety (MM) does not interfere with,obstruct, reduce the ability of, prevent, inhibit, or compete with thetarget binding moiety (TBM) for binding to its target after theactivatable antibody has been activated (e.g., activated by treatmentwith one or more proteases that cleave within the cleavable moiety (CM),activated by a change in pH (increased or decreased), activated by atemperature shift (increased or decreased), activated after beingcontacted with a second molecule (such as an enzyme or a proteinligand), etc.). In some embodiments, the masking moiety (MM) does notinterfere with, obstruct, reduce the ability of, prevent, inhibit, orcompete with the target binding moiety (TBM) for binding its targetafter the cleavable moiety (CM) has been cleaved by one or moreproteases that cleave within the cleavable moiety (CM). In someembodiments, the masking moiety (MM) has a masking efficiency of at mostabout 1.75 (e.g., at most about 1.75, at most about 1.5, at most about1.4, at most about 1.3, at most about 1.2, at most about 1.1, at mostabout 1.0, at most about 0.9, at most about 0.8, at most about 0.7, atmost about 0.6, or at most about 0.5, etc.) after activation (e.g., therelative affinity of the activatable antibody after activation ascompared to the affinity of a parental antibody).

In some embodiments, an activatable antibody of the present disclosure:contains a masking moiety (MM) comprising a pair of cysteine residues atfixed positions to ensure that the activatable antibodies haveconstrained conformations, and/or harbor few or no chemically labileresidues (such as methionine or tryptophan). Advantageously, theinclusion of a pair of cysteine residues at fixed positions ensured thatthe activatable antibodies had constrained conformations, tending toexhibit increased binding affinity and/or specificity. Furthermore,activatable antibodies of the present disclosure included maskingmoieties with few to no unfavorable residues for manufacturingprocesses, such as methionine or tryptophan.

In some embodiments, activatable antibodies of the present disclosureare context-dependent (e.g., are activated (are only capable of bindingtheir targets) in certain contexts (such as in the protease-rich tumormicroenvironment)). In some embodiments, the activatable antibodies ofthe present disclosure provide improved safety over more traditional,non-activatable antibodies (e.g., show reduced toxicity, do not inducesignificant alterations to the weights of many organs, do not alterliver histopathology, hematology, and/or blood biochemistry, etc.). Insome embodiments, activatable antibodies of the present disclosure haveimproved pharmacokinetic properties as compared to more traditional,non-activatable antibodies (e.g., have longer in vivo half-lives).

Anti-CTLA4 Activatable Antibody Activities

In some embodiments, the present disclosure relates to activatableantibodies that bind to human CTLA4 when in active form (e.g., theactivatable antibodies are active after cleavage in the cleavable moiety(e.g., with one or more proteases), but inactive prior to cleavage inthe cleavable moiety (e.g., with one or more proteases)). In someembodiments, the activatable antibodies when in active form have atleast one (e.g., at least one, at least two, at least three, at leastfour, at least five, at least six, at least seven, at least eight, orall nine) of the following functional properties: (a) bind to human,cynomolgus monkey, mouse, rat, and/or dog CTLA4 with a K_(D) of 500 nMor less, e.g., about 10 nM or less; (b) have antagonist activity onhuman CTLA4; (c) do not bind to human PD-1, PD-L1, PD-L2, LAG3, TIM3,B7-H3, CD95, CD120a, OX40, CD40, BTLA, VISTA, ICOS, and/or B7-H4 atconcentration up to 100 nM; (d) are cross-reactive with monkey, mouse,rat, and/or dog CTLA4; (e) induces ADCC effects (e.g., on Tregs); (f)activates human PBMCs (e.g., stimulates secretion of IL-2 and/or IFNγ);(g) are capable of inhibiting tumor cell growth; (h) have therapeuticeffect on a cancer; and (i) inhibit binding of human CTLA4 to human CD80and/or human CD86. Also provided herein are one or more activatableantibodies that compete or cross-compete for binding to human CTLA4 withone or more of the CTLA4-targeting activatable antibodies and/oranti-CTLA4 antibodies described herein.

In some embodiments, the activatable antibodies bind to human,cynomolgus monkey, mouse, rat, and/or dog CTLA4 with a K_(D) of about500 nM or more when in inactive form. In some embodiments, theactivatable antibodies bind to human, cynomolgus monkey, mouse, rat,and/or dog CTLA4 with a K_(D) of about 500 nM or less when in activeform (e.g., about 500 nM or less, about 450 nM or less, about 400 nM orless, about 350 nM or less, about 300 nM or less, about 250 nM or less,about 200 nM or less, about 150 nM or less, about 100 nM or less, about90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM orless, about 50 nM or less, about 40 nM or less, about 30 nM or less,about 25 nM or less, about 20 nM or less, about 10 nM or less, about 1nM or less, about 0.1 nM or less, etc.) In some embodiments, theactivatable antibodies bind to human, cynomolgus monkey, mouse, rat,and/or dog CTLA4 with a K_(D) of about 350 nM or less when in activeform. In some embodiments, the activatable antibodies bind to humanCTLA4 with a K_(D) of about 100 nM or less when in active form. In someembodiments, the activatable antibodies bind to human CTLA4 with a K_(D)of about 50 nM or less when in active form. In some embodiments, theactivatable antibodies bind to human CTLA4 with a K_(D) of about 10 nMor less when in active form. Methods of measuring the K_(D) of anactivatable antibody may be carried out using any method known in theart, including for example, by surface plasmon resonance, an ELISA,isothermal titration calorimetry, a filter binding assay, an EMSA, etc.In some embodiments, the K_(D) is measured by an ELISA (see e.g., theExamples below).

In some embodiments, the activatable antibodies do not have antagonistactivity on human CTLA4 when in inactive form. In some embodiments, theactivatable antibodies have antagonist activity on human CTLA4 when inactive form (e.g., induces ADCC effects (such as against Tregs),activates PBMCs (such as by activating, inducing, and/or stimulatingIL-2 and/or IFNγ secretion), bocks binding of human CTLA4 to human CD80and/or human CD86, etc.). In some embodiments, the activatableantibodies repress one or more activities of human CTLA4 when in activeform (e.g., repress one or more activities of human CTLA4 when a cell(such as a human cell) expressing human CTLA4 is contacted by anactivatable antibody).

In some embodiments, when in inactive form, the activatable antibodiesare not cross-reactive with monkey (e.g., cynomolgus monkey), mouse,rat, and/or dog CTLA4. In some embodiments, when in active form, theactivatable antibodies are cross-reactive with monkey (e.g., cynomolgusmonkey), mouse, rat, and/or dog CTLA4. In some embodiments, when inactive form, the activatable antibodies are cross-reactive with monkeyCTLA4. In some embodiments, when in active form, the activatableantibodies are cross-reactive with mouse CTLA4. In some embodiments,when in active form, the activatable antibodies are cross-reactive withrat CTLA4. In some embodiments, when in active form, the activatableantibodies are cross-reactive with dog CTLA4. In some embodiments, whenin active form, the activatable antibodies are cross reactive withmonkey and mouse CTLA4; monkey and rat CTLA4; monkey and dog CTLA4;mouse and rat CTLA4; mouse and dog CTLA4; rat and dog CTLA4; monkey,mouse, and rat CTLA4; monkey, mouse, and dog CTLA4; monkey, rat, and dogCTLA4; mouse, rat, and dog CTLA4; or monkey, mouse, rat, and dog CTLA4.In some embodiments, when in active form, the activatable bindingpolypeptides are cross-reactive at about 350 nM (e.g., at about 1 nM, atabout 10 nM, at about 25 nM, at about 50 nM, at about 75 nM, at about100 nM, at about 150 nM, at about 200 nM, at about 250 nM, at about 300nM, at about 350 nM). Methods of measuring cross-reactivity are known inthe art, including, without limitation, surface plasmon resonance, anELISA, isothermal titration calorimetry, a filter binding assay, anEMSA, etc.

In some embodiments, the activatable antibodies do not induce ADCCeffects (e.g., against CTLA4-expressing human cells such as Tregs) whenin inactive form. In some embodiments, the activatable antibodies havereduced ADCC effects (e.g., against CTLA4-expressing human cells such asTregs) when in inactive form as compared to a control bindingpolypeptide (e.g., a parental antibody). In some embodiments, theactivatable antibodies induce ADCC effects (e.g., againstCTLA4-expressing such as Tregs) when in active form. Methods ofmeasuring ADCC effects (e.g., in vitro methods) are known in the art,including, without limitation, via the methods described in the Examplesbelow. In some embodiments, when in inactive form, the activatableantibodies induce ADCC effects by less than about 10% (e.g., induce ADCCby less than about 10%, less than about 5%, less than about 1%, etc.)relative to a control (e.g., a parental antibody). In some embodiments,when in active form, the activatable antibodies induce ADCC effects bymore than about 10% (e.g., induce ADCC by more than about 10%, more thanabout 15%, more than about 20%, more than about 25%, more than about30%, more than about 35%, more than about 40%, etc.) relative to acontrol (e.g., an isotype control).

In some embodiments, the activatable antibodies are capable ofinhibiting tumor cell growth and/or proliferation. In some embodiments,the tumor cell growth and/or proliferation is inhibited by at leastabout 5% (e.g., at least about 5%, at least about 10%, at least about20%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, or at least about 99%) when contacted with the activatableantibodies relative to corresponding tumor cells not contacted with theactivatable antibodies (or relative to corresponding tumor cellscontacted with an isotype control antibody). In some embodiments, theactivatable antibodies are capable of reducing tumor volume in a subjectwhen the subject is administered the activatable antibodies. In someembodiments, the activatable antibodies are capable of reducing tumorvolume in a subject by at least about 5% (e.g., at least about 5%, atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, or at least about 99%) relative tothe initial tumor volume in the subject (e.g., prior to administrationof the activatable antibodies; as compared to a corresponding tumor in asubject administered an isotype control antibody). Methods of monitoringtumor cell growth/proliferation, tumor volume, and/or tumor inhibitionare known in the art, including, for example, via the methods describedin the Examples below.

In some embodiments, the activatable antibodies have therapeutic effecton a cancer. In some embodiments, the activatable antibodies reduce oneor more signs or symptoms of a cancer. In some embodiments, a subjectsuffering from a cancer goes into partial or complete remission whenadministered the activatable antibodies.

In some embodiments, the present disclosure provides isolatedactivatable antibodies that, when in active form, compete orcross-compete for binding to human CTLA4 with an antibody comprising: a)an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 23; an HVR-H2comprising the amino acid sequence of SEQ ID NO: 35; and an HVR-H3comprising the amino acid sequence of SEQ ID NO: 45; and/or b) an HVR-L1comprising the amino acid sequence of SEQ ID NO: 58; an HVR-L2comprising the amino acid sequence of SEQ ID NO: 66; and an HVR-L3comprising the amino acid sequence of SEQ ID NO: 75. In someembodiments, the present disclosure provides isolated activatableantibodies that, when in active form, compete or cross-compete forbinding to human CTLA4 with an antibody comprising: a) a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 87;and/or b) a light chain variable region comprising the amino acidsequence of SEQ ID NO: 100. The ability of an activatable antibody tocompete or cross-compete for binding with an antibody can be determinedusing standard binding assays known in the art, such as BIAcoreanalysis, ELISA assays, or flow cytometry. For example, one can allow anantibody (e.g., as described above) to bind to human CTLA4 undersaturating conditions and then measure the ability of the testactivatable antibody (when in active form) to bind to the CTLA4. If thetest activatable antibody is able to bind to the CTLA4 at the same timeas the antibody, then the test activatable antibody binds to a differentepitope then the antibody. However, if the test activatable antibody isnot able to bind to the CTLA4 at the same time, then the testactivatable antibody binds to the same epitope, an overlapping epitope,or an epitope that is in close proximity to the epitope bound by theantibody. This experiment can be performed using various methods, suchas ELISA, RIA, FACS or surface plasmon resonance.

In some embodiments, the activatable antibodies (when in inactive form)do not inhibit the binding between CTLA4 and one or more of its bindingpartners (e.g., human CTLA4 and human CD80, human CTLA4 and human CD86).In some embodiments, the activatable antibodies (when in active form)inhibit the binding between CTLA4 and one or more of its bindingpartners (e.g., human CTLA4 and human CD80, human CTLA4 and human CD86).In some embodiments, the activatable antibodies inhibit the bindingbetween CTLA4 and its ligand in vitro. In some embodiments, theactivatable antibodies have a half maximal inhibitory concentration(IC₅₀) of about 500 nM or less (e.g., about 500 nM or less, about 400 nMor less, about 300 nM or less, about 200 nM or less, about 100 nM orless, about 50 nM or less, about 25 nM or less, about 10 nM or less,about 1 nM or less, etc.) for inhibiting binding of CTLA4 to CD80 and/orCD86. In some embodiments, the activatable antibodies have a halfmaximal inhibitory concentration (IC₅₀) of about 100 nM or less forinhibiting binding of CTLA4 to CD80 and/or CD86. In some embodiments,the activatable antibodies completely inhibit binding of human CTLA4 toCD80 and/or CD86 when provided at a concentration of about 100 nM orgreater (e.g., about 100 nM or greater, about 500 nM or greater, about 1μM or greater, about 10 μM or greater, etc.). As used herein, the term“complete inhibiting” or “completely inhibits” refers to the activatableantibody's ability to reduce binding between a first protein and asecond protein by at least about 80% (e.g., at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 99%,etc.). Methods of measuring the ability of an a polypeptide to inhibitbinding of a first protein (e.g., human CTLA4) and a second protein(e.g., human CD80 or human CD86) are known in the art, including,without limitation, via BIAcore analysis, ELISA assays, and flowcytometry.

Masking Moieties (MMs)

In some embodiments, the present disclosure relates to activatableantibodies comprising a masking moiety (MM). In some embodiments, themasking moiety (MM) comprises an amino acid sequence according toFormula (XVIII): X_(m)CX_(n)CZ_(o) (SEQ ID NO: 134), where m is from2-10, n is from 3-10, and o is from 1-10, where each X is independentlyan amino acid selected from the group consisting of A, C, D, E, F, G, H,I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and where each Z isindependently an amino acid selected from the group consisting of D, A,Y, S, T, N, I, L, F, V, H, and P. In some embodiments, X is not W, M,and/or C. In some embodiments, each X in X_(m) of formula (XVIII) isindependently an amino acid selected from the group consisting of D, A,Y, S, T, N, I, L, F, V, H, and P and/or each X in X_(n) of formula(XVIII) is independently an amino acid selected from the groupconsisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In someembodiments, the MM comprises a polypeptide encoded by a polynucleotidesequence according to Formula (XX): (NNK)_(m)TGY(NNK)_(n)TGY(NHC)_(o)(SEQ ID NO: 136), wherein each N is independently A, G, T, or C, whereineach K is independently T or G, wherein each Y is independently T or C,and wherein each H is independently A, T, or C.

In some embodiments, the masking moiety (MM) comprises an amino acidsequence according to Formula (XIX): Z_(m)CZ_(n)CZ_(o) (SEQ ID NO: 135),where m is from 2-10, n is from 3-10, and o is from 1-10, and each Z isindependently an amino acid selected from the group consisting of D, A,Y, S, T, N, I, L, F, V, H, and P.

In some embodiments, m is from 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3,3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10,5-9, 5-8, 5-7, 5-6, 6-10, 6- 9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or9-10. In some embodiments, m is from 6-8. In some embodiments, m is 3,4, 5, 6, 7, 8, 9, or 10. In some embodiments, m is 6.

In some embodiments, n is from 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10,4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7,7-10, 7-9, 7-8, 8-10, 8-9, or 9-10. In some embodiments, n is from 6-8.In some embodiments, n is 3, 4, 5, 6, 7, 8, 9, or 10. In someembodiments, n is 6. In some embodiments, n is 8.

In some embodiments, o is from 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3,1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6,3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10,6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10. In some embodiments,o is from 1-2. In some embodiments, o is 1, 2, 3, 4, 5, 6, 7, 8, 9, or10. In some embodiments, o is 2.

In some embodiments, the masking moiety (MM) comprises an amino acidsequence according to Formula (XXI): Z₆CX₆CZ₂ (SEQ ID NO: 137), whereeach X is independently an amino acid selected from the group consistingof A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, andwhere each Z is independently an amino acid selected from the groupconsisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

In some embodiments, the masking moiety (MM) comprises an amino acidsequence according to Formula (XXII): Z₆CX₈CZ₂ (SEQ ID NO: 138), whereeach X is independently an amino acid selected from the group consistingof A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, andwhere each Z is independently an amino acid selected from the groupconsisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

In some embodiments, the first peptide (FP) comprises an amino acidsequence according to Formula (XXIII): (Z₆)C(Z₆)C(Z₂) (SEQ ID NO: 139),where each Z is independently an amino acid selected from the groupconsisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

In some embodiments, the masking moiety (MM) comprises an amino acidsequence according to Formula (XXIV): (Z₆)C(Z₈)C(Z₂) (SEQ ID NO: 140),where each Z is independently an amino acid selected from the groupconsisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In someembodiments, an activatable antibody comprises a masking moiety (MM)comprising a sequence selected from the group consisting ofX_(m)CPDHPYPCXX (SEQ ID NO:181), X_(m)CDAFYPYCXX (SEQ ID NO:182),X_(m)CDSHYPYCXX (SEQ ID NO:183), and X_(m)CVPYYYACXX (SEQ ID NO:184),where m is from 2-10, and where each X is independently an amino acidselected from the group consisting of A, C, D, E, F, G, H, I, K, L, M,N, P, Q, R, S, T, V, W, and Y. In some embodiments, an activatableantibody comprises a masking moiety (MM) comprising the sequenceEVGSYNFVADSCPDHPYPCSA (SEQ ID NO:189), EVGSYIVHHSDCDAFYPYCDS (SEQ IDNO:190), EVGSYYSAYPACDSHYPYCNS (SEQ ID NO:191), EVGSYPNPSSDCVPYYYACAY(SEQ ID NO:192), EVGSYYSAYPACDSHYPYCQS (SEQ ID NO:193),EVGSYPQPSSDCVPYYYACAY (SEQ ID NO:195), or EVGSYPNPASDCVPYYYACAY (SEQ IDNO:196). In some embodiments, the MM comprises the sequence ofEDCVPYYYACAY (SEQ ID NO:213), EVGSSDCVPYYYACAY (SEQ ID NO:214),EDCDAFYPYCDS (SEQ ID NO:215), or EVGHSDCDAFYPYCDS (SEQ ID NO:216).

In some embodiments, the masking moiety (MM) comprises an amino acidsequence selected from NFVADSCPDHPYPCSA (SEQ ID NO: 141),IVHHSDCDAFYPYCDS (SEQ ID NO: 142), YSAYPACDSHYPYCNS (SEQ ID NO: 143),PNPSSDCVPYYYACAY (SEQ ID NO: 144), YSAYPACDSHYPYCQS (SEQ ID NO: 145),PQPSSDCVPYYYACAY (SEQ ID NO: 146), and PNPASDCVPYYYACAY (SEQ ID NO:147).

In some embodiments, any of the masking moieties (MMs) described hereinmay further comprise one or more additional amino acid sequences (e.g.,one or more polypeptide tags). Examples of suitable additional aminoacid sequence may include, without limitation, purification tags (suchas his-tags, flag-tags, maltose binding protein andglutathione-S-transferase tags), detection tags (such as tags that maybe detected photometrically (e.g., red or green fluorescent protein,etc.)), tags that have a detectable enzymatic activity (e.g., alkalinephosphatase, etc.), tags containing secretory sequences, leadersequences, and/or stabilizing sequences, protease cleavage sites (e.g.,furin cleavage sites, TEV cleavage sites, Thrombin cleavage sites), andthe like. In some embodiments, the one or more additional amino acidsequences are at the N-terminus of the masking moiety (MM). In someembodiments, the additional amino acid sequence comprises or consists ofthe sequence EVGSY (SEQ ID NO: 148).

In some embodiments, the masking moiety binds to the target bindingmoiety (TBM) and inhibits the activatable antibody from binding to itstarget before activation (e.g., before treatment with one or moreproteases that cleave within the cleavable moiety (CM), beforeundergoing a (local) change in pH (increased or decreased), before atemperature shift (increased or decreased), before being contacted witha second molecule (such as a small molecule or a protein ligand), etc.),but does not bind to the TBM and/or inhibit the activatable antibodyfrom binding to its target after activation (e.g., after treatment withone or more proteases that cleave within the cleavable moiety (CM),after undergoing a (local) change in pH (increased or decreased), aftera temperature shift (increased or decreased), after being contacted witha second molecule (such as a small molecule or a protein ligand), etc.).In some embodiments, the masking moiety (MM) inhibits binding of anactivatable antibody to its target when the CM is not cleaved, but doesnot inhibit binding of the activatable antibody to its target when theCM is cleaved. In some embodiments, the masking moiety (MM) has adissociation constant for binding to the TBM that is greater (e.g., atleast about 1.5-fold greater, at least about 2-fold greater, at leastabout 2.5-fold greater, at least about 3-fold greater, at least about3.5-fold greater, at least about 4-fold greater, at least about 4.5-foldgreater, at least about 5-fold greater, at least about 10-fold greater,at least about 100-fold greater, at least about 500-fold greater, etc.)than the dissociation constant of the activatable antibody for itstarget (when in active form).

Cleavable Moieties (CMs)

In some embodiments, the present disclosure relates to activatableantibodies comprising a cleavable moiety (CM). In some embodiments, thecleavable moiety (CM) is cleaved and/or disrupted by treatment with oneor more proteases that cleave within the cleavable moiety (CM), by achange in pH (increased or decreased), by a temperature shift (increasedor decreased), and/or by contact with a second molecule (such as a smallmolecule or a protein ligand), etc.)

In some embodiments, the cleavable moiety (CM) comprises at least afirst cleavage site (CS₁) (e.g., a first protease cleavage site). Insome embodiments, the first cleavage site is a first protease cleavagesite. Any suitable protease cleavage site recognized and/or cleaved byany protease (e.g., a protease that is known to be co-localized with atarget of an activatable antibody comprising the CM) known in the artmay be used, including, for example, a protease cleavage site recognizedand/or cleaved by urokinase-type plasminogen activator (uPA); matrixmetalloproteinases (e.g., MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9,MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19,MMP-20, MMP-23, MMP-24, MMP-26, and/or MMP-27); Tobacco Etch Virus (TEV)protease; plasmin; Thrombin; PSA; PSMA; ADAMS/ADAMTS (e.g., ADAM 8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAMDEC1, ADAMTS1, ADAMTS4,and/or ADAMTS5); caspases (e.g., Caspase-1, Caspase-2, Caspase-3,Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9,Caspase-10, Caspase-11, Caspase-12, Caspase-13, and/or Caspase-14);aspartate proteases (e.g., RACE and/or Renin); aspartic cathepsins(e.g., Cathepsin D and/or Cathepsin E); cysteine cathepsins (e.g.,Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L, Cathepsin S,Cathepsin V/L2, and/or Cathepsin X/Z/P); cysteine proteinases (e.g.,Cruzipain, Legumain, and/or Otubain-2); KLKs (e.g., KLK4, KLK5, KLK6,KLK7, KLK8, KLK10, KLK11, KLK13, and/or KLK14); metallo proteainases(e.g., Meprin, Neprilysin, PSMA, and/or BMP-1); serine proteases (e.g.,activated protein C, Cathepsin A, Cathepsin G, Chymase, and/orcoagulation factor proteases (such as FVIIa, FIXa, FXa, FXIa, FXIIa));elastase; granzyme B; guanidinobenzoatase; HtrA1; human neutrophilelastase; lactoferrin; marapsin; NS3/4A; PACE4; tPA; tryptase; type IItransmembrane serine proteases (TTSPs) (e.g., DESC1, DPP-4, FAP, Hepsin,Matriptase-2, MT-SP1/Matriptase, TMPRSS2, TMPRSS3 and/or TMPRSS4); etc.In some embodiments, the first protease cleavage site is a cleavage sitefor a protease selected from uPA, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9,MMP-14, TEV protease, plasmin, Thrombin, Factor X, PSA, PSMA, CathepsinD, Cathepsin K, Cathepsin S, ADAM10, ADAM12, ADAMTS, Caspase-1,Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7,Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13,Caspase-14, and TACE. In some embodiments, the first protease cleavagesite is a cleavage site for a protease selected from uPA, MMP-2, MMP-9,and/or TEV protease. In some embodiments, the protease cleavagecomprises an amino acid sequence selected from SGRSA (SEQ ID NO: 149),PLGLAG (SEQ ID NO: 150), and ENLYFQG (SEQ ID NO: 151).

In some embodiments, an activatable antibody comprises a masking moiety(MM) and a cleavable moiety (CM) comprising an amino acid sequenceaccording to Formula (XXV): EVGSY(Z₆)C(Z₆)C(Z₂)SGRSA (SEQ ID NO: 152),where each Z is independently an amino acid selected from D, A, Y, S, T,N, I, L, F, V, H, and P.

In some embodiments, an activatable antibody comprises a masking moiety(MM) and a cleavable moiety (CM) comprising an amino acid sequenceaccording to Formula (XXVI): EVGSY(Z₆)C(X₆)C(Z₂)SGRSA (SEQ ID NO: 153),where each X is independently an amino acid selected from A, C, D, E, F,G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and where each Z isindependently an amino acid selected from the group consisting of D, A,Y, S, T, N, I, L, F, V, H, and P.

In some embodiments, an activatable antibody comprises a masking moiety(MM) and a cleavable moiety (CM) comprising an amino acid sequenceaccording to Formula (XXVII): EVGSY(Z₆)C(Z₈)C(Z₂)SGRSA (SEQ ID NO: 154),where each Z is independently an amino acid selected from D, A, Y, S, T,N, I, L, F, V, H, and P.

In some embodiments, an activatable antibody comprises a masking moiety(MM) and a cleavable moiety (CM) comprising an amino acid sequenceaccording to Formula (XXVIII): EVGSY(Z₆)C(X₈)C(Z₂)SGRSA (SEQ ID NO:155), where each X is independently an amino acid selected from thegroup consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,V, W, and Y, and wherein each Z is independently an amino acid selectedfrom the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

In some embodiments, the cleavable moiety (CM) further comprises a firstlinker (L₁). In some embodiments, the first linker (L₁) is C-terminal tothe first cleavage site (CS₁) (e.g., a first protease cleavage site). Insome embodiments, the cleavable moiety (CM) comprises a structure, fromN-terminus to C-terminus, of: (CS₁)-L₁.

Any suitable linker (e.g., a flexible linker) known in the art may beused, including, for example: glycine polymers (G)n, where n is aninteger of at least 1 (e.g., at least one, at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, atleast 10, etc.); glycine-serine polymers (GS)n, where n is an integer ofat least 1 (e.g., at least one, at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10,etc.) such as GGGGS (SEQ ID NO: 156), SGGS (SEQ ID NO: 157), GGSG (SEQID NO: 158), GGSGG (SEQ ID NO: 159), GSGSG (SEQ ID NO: 160), GSGGG (SEQID NO: 161), GGGSG (SEQ ID NO: 162), and/or GSSSG (SEQ ID NO: 163));glycine-alanine polymers; alanine-serine polymers; and the like. Linkersequences may be of any length, such as from about 1 amino acid (e.g.,glycine or serine) to about 20 amino acids (e.g., 20 amino acid glycinepolymers or glycine-serine polymers), about 1 amino acid to about 15amino acids, about 3 amino acids to about 12 amino acids, about 4 aminoacids to about 10 amino acids, about 5 amino acids to about 9 aminoacids, about 6 amino acids to about 8 amino acids, etc. In someembodiments, the linker is any of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In someembodiments, the linker comprises an amino acid sequence selected fromSEQ ID NOS: 159-163. In some embodiments, the linker comprises an aminoacid sequence of SEQ ID NO: 156 or 157.

In some embodiments, the cleavable moiety (CM) further comprises atleast a second cleavage site (e.g., at least a second, at least a third,at least a fourth, at least a fifth, etc.). In some embodiments, thecleavable moiety (CM) further comprises a second cleavage site (CS₂). Insome embodiments, the second cleavage site is a second protease cleavagesite. The second protease cleavage site may be any suitable proteasecleavage site recognized and/or cleaved by any of the proteasesdescribed above. In some embodiments, the first (CS₁) and second (CS₂)cleavage sites are protease cleavage sites recognized and/or cleaved bythe same protease. In some embodiments, the first (CS₁) and second (CS₂)cleavage sites are protease cleavage sites recognized and/or cleaved bydifferent proteases (e.g., the first protease cleavage site isrecognized and/or cleaved by uPA, and the second protease cleavage siteis recognized and/or cleaved by MMP-2; the first protease cleavage siteis recognized and/or cleaved by uPA, and the second protease cleavagesite is recognized and/or cleaved by MMP-9; the first protease cleavagesite is recognized and/or cleaved by uPA, and the second proteasecleavage site is recognized and/or cleaved by TEV protease; etc.). Insome embodiments, the at least second cleavage site (CS₂) is C-terminalto the first linker (L₁). In some embodiments, the cleavable moiety (CM)comprises a structure, from N-terminus to C-terminus, of:(CS₁)-L₁-(CS₂).

In some embodiments, the cleavable moiety (CM) further comprises atleast a second linker (e.g., at least a second, at least a third, atleast a fourth, at least a fifth, etc.). In some embodiments, thecleavable moiety (CM) further comprises a second linker (L₂). The secondlinker (L₂) may be any suitable linker described above. In someembodiments, the second linker comprises an amino acid sequence selectedfrom SEQ ID NO: 156-163. In some embodiments, the first (L₁) and second(L₂) linkers are the same (e.g., both linkers comprise the sequence ofSEQ ID NO: 156 or 157). In some embodiments, the first (L₁) and second(L₂) linkers are different (e.g., the first linker (L₁) comprises theamino acid sequence of SEQ ID NO: 156, and the second linker (L₂)comprises the amino acid sequence of SEQ ID NO: 157, etc.). In someembodiments, the at least second linker (L₂) is C-terminal to the secondcleavage site (CS₂). In some embodiments, the cleavable moiety (CM)comprises a structure, from N-terminus to C-terminus, of:(CS₁)-L₁-(CS₂)-L₂.

Exemplary MM-CM Sequences

In some embodiments, an activatable antibody of the present disclosurecomprises the structure, from N-terminus to C-terminus, of:(FP)-(PCS₁)-L₁-(PCS₂)-L₂. In some embodiments, an activatable antibodycomprises an amino acid sequence according to Formula (XXIX),EVGSYX₁X₂X₃X₄X₅X₆CX₇X₈X₉X₁₀X₁₁X₁₂CX₁₃X₁₄SGRSAGGGGTENLYFQGSGGS (SEQ IDNO: 164), where X1 is A, D, I, N, P, or Y, X2 is A, F, N, S, or V, X3 isA, H, L, P, S, V, or Y, X4 is A, H, S, or Y, X5 is A, D, P, S, V, or Y,X6 is A, D, L, S, or Y, X7 is D, P, or V, X8 is A, D, H, P, S, or T, X9is A, D, F, H, P, or Y, X10 is L, P, or Y, X11 is F, P, or Y, X12 is A,P, S, or Y, X13 is A, D, N, S, T, or Y, and X14 is A, S, or Y. In someembodiments, an activatable antibody of the present disclosure comprisesthe amino acid sequence of: EVGSYDALHYACPPDYYACYYSGRSAGGGGTENLYFQGSGGS(SEQ ID NO: 165); EVGSYNSYHAYCPHPLYPCTASGRSAGGGGTENLYFQGSGGS (SEQ ID NO:166); EVGSYASSAVLCVTAYFSCNSSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 167);EVGSYNFVADSCPDHPYPCSASGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 168);EVGSYNFVADSCPDHPYPCSASGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 169);EVGSYIVHHSDCDAFYPYCDSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 170);EVGSYIVHHSDCDAFYPYCDSSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 171);EVGSYYSAYPACDSHYPYCNSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 172);EVGSYYSAYPACDSHYPYCNSSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 173);EVGSYPNPSSDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 174);EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 175);EVGSYYSAYPACDSHYPYCQSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 176);EVGSYYSAYPACDSHYPYCNSAGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 177);EVGSYPQPSSDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 178); and/orEVGSYPNPASDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 179). In someembodiments, a polypeptide of the present disclosure comprises thestructure, from N-terminus to C-terminus, of:(FP)-(PCS₁)-L₁-(PCS₂)-L₂-(TBM).

In some embodiments, an activatable antibody comprises an amino acidsequence SGRSAGGGGTENLYFQGSGGS (SEQ ID NO:220), SGRSAGGGGTPLGLAGSGGS(SEQ ID NO:221), or SGRSAPLGLA (SEQ ID NO:222). In some embodiments, anactivatable antibody comprises the sequence of EV(Zn)C(X₈)C(Z₂)SGRSA(SEQ ID NO:217), EDC(Z₆)C(Z₂)SGRSA (SEQ ID NO:218), or EDC(Z₆)C(Z₂)PLGLA(SEQ ID NO:219), where each X is independently an amino acid selectedfrom the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, and Y, wherein n is 1-11 and wherein each Z isindependently an amino acid selected from the group consisting of D, A,Y, S, T, N, I, L, F, V, H, and P.

Target Binding Moieties (TBMs)

In some embodiments, the present disclosure relates to activatableantibodies comprising a target binding moiety (TBM). In someembodiments, the target binding moiety (TBM) comprises an antibody lightchain variable region and/or an antibody heavy chain variable region. Insome embodiments, the target binding moiety (TBM) comprises an antibodylight chain variable region. In some embodiments, the target bindingmoiety (TBM) comprises an antibody heavy chain variable region. In someembodiments, the target binding moiety (TBM) comprises an antibody lightchain variable region and an antibody heavy chain variable region.

In some embodiments, the target binding moiety (TBM) comprises a fulllength antibody light chain and/or a full length antibody heavy chain.The antibody light chain may be a kappa or lambda light chain. Theantibody heavy chain may be in any class, such as IgG, IgM, IgE, IgA, orIgD. In some embodiments, the antibody heavy chain is in the IgG class,such as IgG1, IgG2, IgG3, or IgG4 subclass. An antibody heavy chaindescribed herein may be converted from one class or subclass to anotherclass or subclass using methods known in the art.

Any one or more of the target binding moieties (TBMs) described hereinmay incorporate: any of the HVR sequences described herein (e.g., one,two, or three of the heavy chain variable region HVR sequences, and/orone, two, or three of the light chain variable region HVR sequences asshown in Table A above); any of the heavy chain variable regionsequences and/or light chain variable region sequences described herein(e.g., a heavy chain variable region sequence and/or a light chainvariable region sequence as shown in Table B above); and/or any of anyof the antibodies described herein.

In some embodiments, the target binding moiety (TBM) comprises asequence of one or more of the anti-CTLA4 antibodies described herein,including antibodies described with reference to specific amino acidsequences of HVRs, variable regions (VL, VH), and/or light and heavychains (e.g., IgG1, IgG2, IgG4). In some embodiments, the target bindingmoiety (TBM) comprises an antibody light chain variable regioncomprising an HVR-L1 comprising the amino acid sequence RASQSVRGRFLA(SEQ ID NO: 58), an HVR-L2 comprising the amino acid sequence DASNRATGI(SEQ ID NO: 66), and/or an HVR-L3 comprising the amino acid sequenceYCQQSSSWPPT (SEQ ID NO: 75). In some embodiments, the target bindingmoiety (TBM) comprises an antibody light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 100 or a sequencehaving at least 90% (e.g., 95%, 96%, 97%, 98% or 99%) sequence identityto the sequence of SEQ ID NO:100. In some embodiments, the targetbinding moiety (TBM) comprises an antibody heavy chain variable regioncomprising an HVR-H1 comprising the amino acid sequence YSISSGYHWSWI(SEQ ID NO: 23), an HVR-H2 comprising the amino acid sequenceLARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35), and/or an HVR-H3 comprising theamino acid sequence ARSYVYFDY (SEQ ID NO: 45). In some embodiments, thetarget binding moiety (TBM) comprises an antibody heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 87 or a sequencehaving at least 90% (e.g., 95%, 96%, 97%, 98% or 99%) sequence identityto the sequence of SEQ ID NO:87. In some embodiments, the target bindingmoiety (TBM) comprises: a) an antibody light chain variable regioncomprising an HVR-L1 comprising the amino acid sequence RASQSVRGRFLA(SEQ ID NO: 58), an HVR-L2 comprising the amino acid sequence DASNRATGI(SEQ ID NO: 66), and/or an HVR-L3 comprising the amino acid sequenceYCQQSSSWPPT (SEQ ID NO: 75); and b) an antibody heavy chain variableregion comprising an HVR-H1 comprising the amino acid sequenceYSISSGYHWSWI (SEQ ID NO: 23), an HVR-H2 comprising the amino acidsequence LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35), and/or an HVR-H3comprising the amino acid sequence ARSYVYFDY (SEQ ID NO: 45). In someembodiments, the target binding moiety (TBM) comprises an antibody lightchain variable region comprising the amino acid sequence of SEQ ID NO:100, and an antibody heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 87.

Activatable Binding Polypeptide Properties

In some embodiments, an activatable binding polypeptide (i.e.,activatable antibody) of the present disclosure comprises: (a) a maskingmoiety (MM), (b) a cleavable moiety, and (c) a target binding moiety. Insome embodiments, the masking moiety (MM) binds to the target bindingmoiety (TBM) of the activatable antibody and reduces or inhibits bindingof the activatable binding moiety to CTLA4 (e.g., human CTLA4), ascompared to the binding of a corresponding binding polypeptide lackingthe masking moiety to CTLA4 (e.g., human CTLA4) and/or as compared tothe binding of a parental antibody to CTLA4 (e.g., human CTLA4).

In some embodiments, an “activatable” binding polypeptides refers to abinding polypeptide that exhibits a first level of binding to CTLA4 whenin an inhibited, masked, and/or uncleaved state, and exhibits a secondlevel of binding to CTLA4 in an uninhibited, unmasked, and/or cleavedstate, where the second level of CTLA4 binding is greater than the firstlevel of CTLA4 binding. In some embodiments, access to CTLA4 by theactivatable binding polypeptide is greater after cleavage within thecleavable moiety (e.g., by one or more proteases).

In some embodiments, an activatable antibody of the present disclosureis generally considered to be an “activatable” binding polypeptide whenbinding affinity of the polypeptide to CTLA4 (e.g., human CTLA4)increases by at least about 2-fold (e.g., at least about 2-fold, atleast about 2.5-fold, at least about 3, at least about 3.5-fold, atleast about 4-fold, at least about 4.5-fold, at least about 5-fold, atleast about 5.5-fold, at least about 6-fold, at least about 6.5-fold, atleast about 7-fold, at least about 7.5-fold, at least about 8-fold, atleast about 8.5-fold, at least about 9-fold, at least about 9.5-fold, atleast about 10-fold, at least about 25-fold, at least about 50-fold, atleast about 75-fold, at least about 100-fold, at least about 250-fold,at least about 500-fold, at least about 750-fold, or at least about1000-fold, or more) after activation of the activatable antibody ascompared to prior to activation of the activatable antibody (e.g., afteractivation by treatment with one or more proteases that cleave withinthe cleavable moiety (CM), after activation by a change in pH (increasedor decreased), after activation by a temperature shift (increased ordecreased), after activation by being contacted with a second molecule(such as a small molecule), etc.). In some embodiments, an activatableantibody of the present disclosure is generally considered “activatable”if the EC₅₀ of the activatable antibody decreases by at least about2-fold (e.g., at least about 2-fold, at least about 2.5-fold, at leastabout 3, at least about 3.5-fold, at least about 4-fold, at least about4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about6-fold, at least about 6.5-fold, at least about 7-fold, at least about7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about9-fold, at least about 9.5-fold, at least about 10-fold, at least about25-fold, at least about 50-fold, at least about 75-fold, at least about100-fold, at least about 250-fold, at least about 500-fold, at leastabout 750-fold, or at least about 1000-fold, or more) after “activation”(e.g., as measured by an ELISA or FACS assay; see the examples below).In some embodiments, an activatable antibody of the present disclosureis generally considered “activatable” if the EC₅₀ of the polypeptidedecreases by at least about 2-fold after treatment with a protease thatcleaves within the cleavable moiety (CM) (e.g., as measured by an ELISAor FACS assay; see the examples below).

In some embodiments, when the masking moiety (MM) is bound to the targetbinding moiety (TBM) of the activatable antibody, the K_(D) of theactivatable antibody for CTLA4 is about 2 (e.g., about 2, about 2.5,about 3, about 3.5 about 4, about 4.5, about 5, about 5.5, about 6,about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5,about 10, about 25, about 50, about 75, about 100, about 250, about 500,about 750, or about 1000 or more) times greater than when the maskingmoiety (MM) is not bound to the target binding moiety (TBM) (e.g., after“activation” of the activatable antibody (such as after proteasetreatment to cleave within the cleavable moiety (CM))) and/or than theK_(D) of the parental antibody for CTLA4. Methods of measuring affinityare known in the art, including, for example, by the methods describedin the Examples below).

In some embodiments, when the masking moiety is bound to the targetbinding moiety of the activatable antibody, the K_(D) of the activatableantibody for CTLA4 is reduced by at least about 25% (e.g., at leastabout 25%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, at leastabout 90%, at least about 95%, at least about 99%) relative to when themasking moiety is not bound to the target binding moiety (e.g., after“activation” of the activatable antibody (such as after proteasetreatment to cleave within the cleavable moiety (CM))) and/or relativeto the K_(D) of the parental antibody for CTLA4. Methods of measuringaffinity are known in the art, including, for example, by the methodsdescribed in the Examples below).

In some embodiments, the masking moiety sterically hinders binding ofthe activatable antibody to CTLA4 and/or allosterically hinders bindingof the activatable antibody to CTLA4. In some embodiments, the maskingmoiety does not comprise an amino acid sequence of a natural bindingpartner of the activatable antibody and/or parental antibody.

In some embodiments, the dissociation constant of the masking moiety forthe target binding moiety is greater than the dissociation constant forthe activatable antibody for CTLA4 (when activated). In someembodiments, the dissociation constant of the masking moiety for thetarget binding moiety is about 2 (e.g., about 2, about 2.5, about 3,about 3.5 about 4, about 4.5, about 5, about 5.5, about 6, about 6.5,about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10,about 25, about 50, about 75, about 100, about 250, about 500, about750, or about 1000 or more) times greater than the dissociation constantfor the activatable antibody for CTLA4 (when activated). In someembodiments, the dissociation constant of the masking moiety for thetarget binding moiety is about equal to the dissociation constant forthe activatable antibody for CTLA4 (when activated).

The activatable antibodies described herein may be further modified. Insome embodiments, the activatable antibodies are linked to an additionalmolecular entity. Examples of additional molecular entities includepharmaceutical agents, peptides or proteins, detection agent or labels,and antibodies.

In some embodiments, an activatable antibody of the present disclosureis linked to a pharmaceutical agent. Examples of pharmaceutical agentsinclude cytotoxic agents or other cancer therapeutic agents, andradioactive isotopes. Specific examples of cytotoxic agents includetaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents also include, for example,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). Examples of radioactive isotopesthat can be conjugated to antibodies for use diagnostically ortherapeutically include, but are not limited to, iodine¹³¹, indium¹¹¹,yttrium⁹⁰ and lutetium¹⁷⁷. Methods for linking a polypeptide to apharmaceutical agent are known in the art, such as using various linkertechnologies. Examples of linker types include hydrazones, thioethers,esters, disulfides and peptide-containing linkers. For furtherdiscussion of linkers and methods for linking therapeutic agents toantibodies see e.g., Saito et al., Adv. Drug Deliv. Rev. 55:199-215(2003); Trail, et al., Cancer Immunol. Immunother. 52:328-337 (2003);Payne, Cancer Cell 3:207-212 (2003); Allen, Nat. Rev. Cancer 2:750-763(2002); Pastan and Kreitman, Curr. Opin. Investig. Drugs 3:1089-1091(2002); Senter and Springer (2001) Adv. Drug Deliv. Rev. 53:247-264.

V. Nucleic Acids, Vectors, Host Cells, and Recombinant Methods ofProducing CTLA4 Antibodies and/or Precision/Context-DependentActivatable Antibodies

Another aspect of the disclosure provides an isolated nucleic acidmolecule that comprises a nucleotide sequence encoding an amino acidsequence of a binding molecule (e.g., an antibody or activatableantibody) provided herein. The amino acid sequence encoded by thenucleotide sequence may be any portion of an antibody, such as an HVR, asequence comprising one, two, or three HVRs, a variable region of aheavy chain, variable region of a light chain, or may be a full-lengthheavy chain or full length light chain. A nucleic acid of the disclosurecan be, for example, DNA or RNA, and may or may not contain intronicsequences. Typically, the nucleic acid is a cDNA molecule.

In some embodiments, the disclosure provides an isolated nucleic acidmolecule that comprises or consists of a nucleotide sequence encoding anamino acid sequence selected from the group consisting of: (1) aminoacid sequence of an HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and/or andHVR-L3 of an illustrative antibody described herein; (2) a variableregion of a heavy chain and/or variable region of a light chain of anillustrative antibody described herein; or (3) a full length heavy chainor full length light chain of an illustrative antibody.

In some embodiments, the nucleic acid molecule comprises or consists ofa nucleotide sequence that encodes an amino acid sequence as set forthin any one of SEQ ID NOS: 18-107.

In some embodiments, the nucleic acid molecule comprises or consists ofa nucleotide sequence described in Table C below.

TABLE C anti-CTLA4 variable region polynucleotide sequences Ab name: VH:VL: TY21585 GAGGTTCAGCTGGTGGAGTCTGGCGGTGG GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGGTGCAGCCAGGGGGCTCACTCCGTTT CCTGTCCGCCTCTGTGGGCGATCGGGTCAGTCCTGTGCAGCTTCCGGATTCACCTTCTCC CCATCACCTGCCGTGCCTCTGAGTCTGTGGACTACGCTATTCACTGGGTGCGTCAGGCC GACTTCTTCGGTATCTCTTTCCTGGCCTGGCCGGGTAAGGGACTCGAGTGGATCGGTATC TATCAACAGAAACCAGGAAAAGCTCCGAATCTCCCCATCTAGCGGTTCTACTAACTACG AGCTTCTGATCTACGACGCCTCTAACCGTCCCAGAAGTTCCAGGGTCGTGTGACTATAA GCCACCGGTATCCCATCTCGCTTCTCTGGAGTCGCGACAATTCGAAAAACACACTGTACC TCCGGTTCCGGGACGGATTTCACTCTGACTACAACTGAACAGCTTAAGAGCTGAGGAC CATCAGCAGTCTGCAGCCGGAAGACTTCACTGCCGTCTATTATTGCGCCAGAGACATTC GCAACTTATTACTGCCAGCACTACACCTCACTCTGGTTCTTCTGGTTACTACTACGGTTT TTCGCCACCAGTGTACACCTTCGGACAGGCGACGTCTGGGGTCAAGGAACACTAGTCA GTACCAAGGTGGAGATCAAACGA CCGTCTCCTCG(SEQ ID NO: 121) (SEQ ID NO: 108) TY21586 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGGATATCCAGTTGACCCAGTCCCCGAGTTC CCTGGTGCAGCCAGGGGGCTCACTCCGTTTCCTGTCCGCCTCTGTGGGCGATCGGGTCA GTCCTGTGCAGCTTCCGGATACTCTATCACCCCATCACCTGCTCTGCCTCTTCTAGCGTG TCTGGTTACTACTGGGCCTGGATTCGTCAGAGCTACGTGTACTGGTATCAACAGAAACC GCCCCGGGTAAGGGCCTCGAGTGGGTGTCTAGGAAAAGCTCCGAAGCTTCTGATCTACG TCCATCTCTGGTTCCGGTTCTACTACCTACTACGCCTCTTCTCTGGAATCTGGTGTGCCA ACGCCGACTCTGTCAAGGGCCGTTTCACTATCTCGCTTCTCTGGATCCGGTTCCGGGAC TAAGTCGCGACAATTCGAAAAACACACTGTGGATTTCACTCTGACCATCAGCAGTCTGC ACCTACAACTGAACAGCTTAAGAGCTGAGAGCCGGAAGACTTCGCAACTTATTACTGC GACACTGCCGTCTATTATTGCGCCAGAGATGTGCAGGGTCTTCAGACCCCTTGGACCTT GGTTTCGGCTACTTCGACTACTGGGGTCAACGGACAGGGTACCAAGGTGGAGATCAAA GGAACACTAGTCACCGTCTCCTCG CGA(SEQ ID NO: 109) (SEQ ID NO: 122) TY21587 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGGATATCCAGTTGACCCAGTCCCCGAGTTC CCTGGTGCAGCCAGGGGGCTCACTCCGTTTCCTGTCCGCCTCTGTGGGCGATCGGGTCA GTCCTGTGCAGCTTCCGGATTCACCTTCTCCCCATCACCTGCCGTGCCTCTCAGGGTATT GACTACGGTATTCACTGGGTGCGTCAGGCCGGCTCTTCCCTGGCTTGGTATCAACAGAA CCGGGTAAGGGCCTCGAGTGGATCGGTGAACCAGGAAAAGCTCCGAAGCTTCTGATCT AATCTACCACTCTGGTTCTACCTACTACTCTACGACGCCTCTAACCGTGCCACCGGTATC CCATCTCTGAAGTCTCGTGTGACTATAAGTCCCATCTCGCTTCTCTGGATCCGGTTCCGG GCGACAATTCGAAAAACACACTGTACCTACGACGGATTTCACTCTGACCATCAGCAGTC AACTGAACAGCTTAAGAGCTGAGGACACTTGCAGCCGGAAGACTTCGCAACTTATTAC GCCGTCTATTATTGCGCCAGAGACGTTGCCTGCCAGCAGTACGACCAATGGCCACCTTG CCTGGTTCTTCTGGTTACTACGACGGTTTCGGACCTTCGGACAGGGTACCAAGGTGGAG ACTTCTGGGGTCAAGGAACACTAGTCACCG ATCAAACGATCTCCTCG (SEQ ID NO: 123) (SEQ ID NO: 110) TY21588GAGGTTCAGCTGGTGGAGTCTGGCGGTGG GATATCCAGTTGACCCAGTCCCCGAGTTCCCTGGTGCAGCCAGGGGGCTCACTCCGTTT CCTGTCCGCCTCTGTGGGCGATCGGGTCAGTCCTGTGCAGCTTCCGGATACTCTATCTCC CCATCACCTGCCGTGCCTCTGAGTCTGTGTCTGGTTACCACTGGGACTGGATTCGTCAG GACTTCTTCGGTAAGTCTTTCCTGCACTGGCCCCGGGTAAGGGCCTCGAGTGGGTGTCT GTATCAACAGAAACCAGGAAAAGCTCCGGGTATCTCTGGTTACGGTGGTTCTACCTACT AAGCTTCTGATCTACGACGCCTCTAACCTACGCCGACTCTGTCAAGGGCCGTTTCACTA GGAAACCGGTGTGCCATCTCGCTTCTCTGTAAGTCGCGACAATTCGAAAAACACACTGT GATCCGGTTCCGGGACGGATTTCACTCTGACCTACAACTGAACAGCTTAAGAGCTGAG ACCATCAGCAGTCTGCAGCCGGAAGACTGACACTGCCGTCTATTATTGCGCCAGACAC TCGCAACTTATTACTGCCAGCAGTCCTACAGTTATTACGGTTCCGGTAATTTCGACTACT TCCTGGCCTCCGACCTTCGGACAGGGTACGGGGTCAAGGAACACTAGTCACCGTCTCCT CAAGGTGGAGATCAAACGA CG (SEQ ID NO: 124)(SEQ ID NO: 111) TY21589 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGGATATCCAGTTGACCCAGTCCCCGAGTTC CCTGGTGCAGCCAGGGGGCTCACTCCGTTTCCTGTCCGCCTCTGTGGGCGATCGGGTCA GTCCTGTGCAGCTTCCGGATTCACCTTCTCCCCATCACCTGCCGTGCCTCTCAGTCTGTG GACTACTGGATTCACTGGGTGCGTCAGGCCAGCAGCCGTTTCCTGGCCTGGTATCAACA CCGGGTAAGGGCCTCGAGTGGATCGGTTGGGAAACCAGGAAAAGCTCCGAAGCTTCTG ATCTCCCCATCTGGCGGTGGTACTAAGTACATCTACGACGCCTCTAACCGTGCCACCGG GCCCAGAAGTTCCAGGGTCGTGTGACTATATATCCCATCTCGCTTCTCTGGATCCGGTTC AGTCGCGACAATTCGAAAAACACACTGTACCGGGACGGATTTCACTCTGACCATCAGCA CTACAACTGAACAGCTTAAGAGCTGAGGAGTCTGCAGCCGGAAGACTTCGCAACTTAT CACTGCCGTCTATTATTGCGCCAGAGGGGCTACTGCCAGCAGTCCTACCCCACCCCTCT TTACGAATTTGACTACTGGGGTCAAGGAACTACCTTCGGACAGGGTACCAAGGTGGAG ACTAGTCACCGTCTCCTCG ATCAAACGA(SEQ ID NO: 112) (SEQ ID NO: 125) TY21580 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGGATATCCAGTTGACCCAGTCCCCGAGTTC CCTGGTGCAGCCAGGGGGCTCACTCCGTTTCCTGTCCGCCTCTGTGGGCGATCGGGTCA GTCCTGTGCAGCTTCCGGATACTCTATCTCCCCATCACCTGCCGTGCCTCTCAGTCTGTG TCTGGTTACCACTGGAGCTGGATTCGTCAGCGCGGCCGTTTCCTGGCCTGGTATCAACA GCCCCGGGTAAGGGCCTCGAGTGGCTGGCGAAACCAGGAAAAGCTCCGAAGCTTCTG CCGGATCGACTGGGACGATGACAAGTACTAATCTACGACGCCTCTAACCGTGCCACCGG CTCTACCTCTCTGAAGTCTCGTCTGACTATATATCCCATCTCGCTTCTCTGGATCCGGTTC AGTCGCGACAATTCGAAAAACACACTGTACCGGGACGGATTTCACTCTGACCATCAGCA CTACAACTGAACAGCTTAAGAGCTGAGGAGTCTGCAGCCGGAAGACTTCGCAACTTAT CACTGCCGTCTATTATTGCGCCAGATCGTACTACTGCCAGCAGTCCTCCTCCTGGCCTCC GTGTACTTCGACTACTGGGGTCAAGGAACAGACCTTCGGACAGGGTACCAAGGTGGAG CTAGTCACCGTCTCCTCG ATCAAACGA(SEQ ID NO: 113) (SEQ ID NO: 126) TY21591 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGGATATCCAGTTGACCCAGTCCCCGAGTTC CCTGGTGCAGCCAGGGGGCTCACTCCGTTTCCTGTCCGCCTCTGTGGGCGATCGGGTCA GTCCTGTGCAGCTTCCGGATTCTCTCTGTCTCCATCACCTGCCGTGCCTCTCAGACCGTG ACCGGCGGTGTGGCTGTGAGCTGGATTCGTTTCTCTCGTTACCTGGCTTGGTATCAACA CAGGCCCCGGGTAAGGGCCTCGAGTGGATGAAACCAGGAAAAGCTCCGAAGCTTCTG CGGTGAAATCTACCACTCTGGTTCTACCTACATCTACGACGCCTCTAACCGTGCCACCGG TACTCTCCATCTCTGAAGTCTCGTGTGACTATATCCCATCTCGCTTCTCTGGATCCGGTTC TAAGTCGCGACAATTCGAAAAACACACTGTCGGGACGGATTTCACTCTGACCATCAGCA ACCTACAACTGAACAGCTTAAGAGCTGAGGTCTGCAGCCGGAAGACTTCGCAACTTAT GACACTGCCGTCTATTATTGCGCCCGTCGTATACTGCCAGCAGTCCTACTACTGGCCACC TCGCCACCGCTACTTACTTCGACTACTGGGTTGGACCTTCGGACAGGGTACCAAGGTG GTCAAGGAACACTAGTCACCGTCTCCTCG GAGATCAAACGA(SEQ ID NO: 114) (SEQ ID NO: 127) TY21686 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGGATATCCAGTTGACCCAGTCCCCGAGTTC CCTGGTGCAGCCAGGGGGCTCACTCCGTTTCCTGTCCGCCTCTGTGGGCGATCGGGTCA GTCCTGTGCAGCTTCCGGATTCTCTCTGTCTCCATCACCTGCCGTGCCTCTCAGGGTGTG ACCGGCGGTGTGGCTGTGGGCTGGATTCGTTCTTCTTACCTGGCCTGGTATCAACAGAA CAGGCCCCGGGTAAGGGCCTCGAGTGGGTACCAGGAAAAGCTCCGAAGCTTCTGATCT GTCTGCTATCTCTGGTTACGGTTCTACTACCACGCCGCCTCTACCTTGCAGTCTGGTGTG TACTACGCCGACTCTGTCAAGGGCCGTTTCCCATCTCGCTTCTCTGGATCCGGTTCCGG ACTATAAGTCGCGACAATTCGAAAAACACAGACGGATTTCACTCTGACCATCAGCAGTC CTGTACCTACAACTGAACAGCTTAAGAGCTTGCAGCCGGAAGACTTCGCAACTTACTAC GAGGACACTGCCGTCTATTATTGCGCCAGATGCCAGCACCACTACGGCACCCCACTGA TTGCCATACTCCGCCTACGCTTTCGACTACTCCTTCGGTCAGGGTACCAAGGTGGAGATC GGGGTCAAGGAACACTAGTCACCGTCTCCT AAACGA CG(SEQ ID NO: 128) (SEQ ID NO: 115) TY21687 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGGATATCCAGTTGACCCAGTCCCCGAGTTC CCTGGTGCAGCCAGGGGGCTCACTCCGTTTCCTGTCCGCCTCTGTGGGCGATCGGGTCA GTCCTGTGCAGCTTCCGGATTCACCTTCTCCCCATCACCTGCCGTGCCTCTCAGTCTGTG GGCTACGCTATTCACTGGGTGCGTCAGGCCGACTTCTACGGTATCTCTTTCCTGGACTG CCGGGTAAGGGCCTCGAGTGGATCGGTATCGTATCAACAGAAACCAGGAAAAGCTCCG ATCTCCCCATCTGGCGGTGGTACTAAGTACAAGCTTCTGATCTACGACGCCTCTAACCG GCCCAGAAGTTCCAGGGTCGTGTGACTATATGCCACCGGTATCCCATCTCGCTTCTCTGG AGTCGCGACAATTCGAAAAACACACTGTACATCCGGTTCCGGGACGGATTTCACTCTGA CTACAACTGAACAGCTTAAGAGCTGAGGACCATCAGCAGTCTGCAGCCGGAAGACTT CACTGCCGTCTATTATTGCGCCAGACACCCACGCAACTTATTACTGCCAGCAGTACGTCT TTCGCCTACTGGGGTCAAGGAACACTAGTCCTTCGCCACCAGAGTACACCTTCGGACAG ACCGTCTCCTCG GGTACCAAGGTGGAGATCAAACGA(SEQ ID NO: 116) (SEQ ID NO: 129) TY21689 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGGATATCCAGTTGACCCAGTCCCCGAGTTC CCTGGTGCAGCCAGGGGGCTCACTCCGTTTCCTGTCCGCCTCTGTGGGCGATCGGGTCA GTCCTGTGCAGCTTCCGGATACACCTTCTCCCATCACCTGCCGTGCCTCTCAGTCTGTG CGGCTACGGTATTCACTGGGTGCGTCAGGCGACTTCGACGGTTTCTCTTTCCTGCACTG CCCGGGTAAGGGCCTCGAGTGGATCGGTGGTATCAACAGAAACCAGGAAAAGCTCCG AAATCTACCACTCTGGTTCTACCTACTACTCAAGCTTCTGATCTACGACGCCTCTTCTCT TCCATCTCTGAAGTCTCGTGTGACTATAAGTGGAATCTGGTGTGCCATCTCGCTTCTCTG CGCGACAATTCGAAAAACACACTGTACCTAGATCCGGTTCCGGGACGGATTTCACTCTG CAACTGAACAGCTTAAGAGCTGAGGACACACCATCAGCAGTCTGCAGCCGGAAGACT TGCCGTCTATTATTGCGCCAGAAGAATTGACTCGCAACTTATTACTGCCAGCAGCGTGAC GCCTTCGACATCTGGGGTCAAGGAACACTATCCTGGCCTTACACCTTCGGACAGGGTAC GTCACCGTCTCCTCG CAAGGTGGAGATCAAACGA(SEQ ID NO: 117) (SEQ ID NO: 130) TY21680 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGGATATCCAGTTGACCCAGTCCCCGAGTTC CCTGGTGCAGCCAGGGGGCTCACTCCGTTTCCTGTCCGCCTCTGTGGGCGATCGGGTCA GTCCTGTGCAGCTTCCGGATACACCTTCTCCCATCACCTGCCGTGCCTCTCAGTCTGTG CGGCTACGCTATTCACTGGGTGCGTCAGGCGACTTCCACGGTAAGTCTTTCCTGCACTG CCCGGGTAAGGGCCTCGAGTGGATCGGTATGTATCAACAGAAACCAGGAAAAGCTCCG CATCTCCCCATCTGGCGGTGGTACTAAGTACAAGCTTCTGATCTACGACGCCTCTTCTCT GCCCAGAAGTTCCAGGGTCGTGTGACTATAGGAATCTGGTGTGCCATCTCGCTTCTCTG AGTCGCGACAATTCGAAAAACACACTGTACGATCCGGTTCCGGGACGGATTTCACTCTG CTACAACTGAACAGCTTAAGAGCTGAGGAACCATCAGCAGTCTGCAGCCGGAAGACT CACTGCCGTCTATTATTGCGCCAGACTCTATTCGCAACTTATTACTGCGAGCAATCCCTG GACGTTGCCTACTGGGGTCAAGGAACACTAGAAGTCCCATTCACCTTCGGACAGGGTAC GTCACCGTCTCCTCG CAAGGTGGAGATCAAACGA(SEQ ID NO: 118) (SEQ ID NO: 131) TY21691 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGGATATCCAGTTGACCCAGTCCCCGAGTTC CCTGGTGCAGCCAGGGGGCTCACTCCGTTTCCTGTCCGCCTCTGTGGGCGATCGGGTCA GTCCTGTGCAGCTTCCGGATTCACCTTCTCCCCATCACCTGCCGTGCCTCTCAGTCTGTG GACTACGCTATTCACTGGGTGCGTCAGGCCGACTTCTACGGTATCTCTTTCCTGCACTGG CCGGGTAAGGGCCTCGAGTGGATCGGTATCTATCAACAGAAACCAGGAAAAGCTCCGA ATCTCCCCATCTGGCGGTTCTACTAAGTACGAGCTTCTGATCTACGACGCCTCTTCTCTG CCCAGAAGTTCCAGGGTCGTGTGACTATAAGAATCTGGTGTGCCATCTCGCTTCTCTGG GTCGCGACAATTCGAAAAACACACTGTACCATCCGGTTCCGGGACGGATTTCACTCTGA TACAACTGAACAGCTTAAGAGCTGAGGACCCATCAGCAGTCTGCAGCCGGAAGACTT ACTGCCGTCTATTATTGCGCCAGACTCGGTTCGCAACTTATTACTGCGTGCAGGCTCTTC ACGGGTACTTCGACGTCTGGGGTCAAGGAAGTTGCCTCTTACCTTCGGACAGGGTACC ACACTAGTCACCGTCTCCTCG AAGGTGGAGATCAAACGA(SEQ ID NO: 119) (SEQ ID NO: 132) TY21692 GAGGTTCAGCTGGTGGAGTCTGGCGGTGGGATATCCAGTTGACCCAGTCCCCGAGTTC CCTGGTGCAGCCAGGGGGCTCACTCCGTTTCCTGTCCGCCTCTGTGGGCGATCGGGTCA GTCCTGTGCAGCTTCCGGATACTCTATCACCCCATCACCTGCCGTGCCTCTCAGTCTATCT TCTGGTCACTACTGGAGCTGGATTCGTCAGCTTCTTACCTGAACTGGTATCAACAGAAA GCCCCGGGTAAGGGCCTCGAGTGGATCGGTCCAGGAAAAGCTCCGAAGCTTCTGATCTA GACATCTCCCACTCTGGTTCTACCTACTACTCGACGCCTCTAACCTGGAAACCGGTGTG CTCAATCTCTGAAGTCTCGTGTGACTATAAGCCATCTCGCTTCTCTGGATCCGGTTCCGG TCGCGACAATTCGAAAAACACACTGTACCTGACGGATTTCACTCTGACCATCAGCAGTC ACAACTGAACAGCTTAAGAGCTGAGGACATGCAGCCGGAAGACTTCGCAACTTACTAC CTGCCGTCTATTATTGCGCGCGTGGTAGTAGTGCCAGCACCACTACGGCACCCCACTGA GACCGGCTACTTCGACTATTGGGGTCAAGGCCTTCGGTCAGGGTACCAAGGTGGAGATC AACACTAGTCACCGTCTCCTCG AAACGA(SEQ ID NO: 120) (SEQ ID NO: 133)

Nucleic acids of the present disclosure may be obtained using anysuitable molecular biology techniques. For antibodies expressed byhybridomas, cDNAs encoding the light and heavy chains of the antibodymade by the hybridoma can be obtained by PCR amplification or cDNAcloning techniques. For antibodies obtained from an immunoglobulin genelibrary (e.g., using phage display techniques), the nucleic acidencoding the antibody can be recovered from the library.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the V_(H)-encodingDNA to another DNA molecule encoding heavy chain constant regions (CH1,CH2 and CH3). The sequences of human heavy chain constant region genesare known in the art (see e.g., Kabat et al. (1991) NIH Publication No.91-3242) and DNA fragments encompassing these regions can be obtained bystandard PCR amplification. The heavy chain constant region can be anIgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. For a Fabfragment heavy chain gene, the V_(H)-encoding DNA can be operativelylinked to another DNA molecule encoding only the heavy chain CH1constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, CL. The sequences of humanlight chain constant region genes are known in the art (see e.g., Kabatet al. (1991) NIH Publication No. 91-3242) and DNA fragmentsencompassing these regions can be obtained by standard PCRamplification. The light chain constant region can be a kappa or lambdaconstant region.

To create an scFv gene, the V_(H)- and V_(L)-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the V_(H) andV_(L) sequences can be expressed as a contiguous single-chain protein,with the V_(L) and V_(H) regions joined by the flexible linker (seee.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc.Natl. Acad. Sci. USA 85:5879-5883 (1988); and McCafferty et al., Nature348:552-554 (1990)).

The present disclosure further provides a vector that comprises anucleic acid molecule described herein. In some embodiments, the vectoris an expression vector or a display vector (e.g., a viral displayvector, a bacterial display vector, a yeast display vector, an insectdisplay vector, a mammalian display vector, etc.). The nucleic acidmolecule may encode a portion of a light chain or heavy chain (such as aCDR or a HVR; a light or heavy chain variable region), a full-lengthlight or heavy chain, polypeptide that comprises a portion orfull-length of a heavy or light chain, or an amino acid sequence of anantibody derivative or antigen-binding fragment. In some embodiments,the vector is an expression vector useful for the expression of abinding molecule, such as an antibody or an antigen binding fragmentthereof. In some embodiments, provided herein are vectors, wherein afirst vector comprises a polynucleotide sequence encoding a heavy chainvariable region as described herein, and a second vector comprises apolynucleotide sequence encoding a light chain variable region asdescribed herein. In some embodiments, a single vector comprisespolynucleotides encoding a heavy chain variable region as describedherein and a light chain variable region as described herein.

To express a binding molecule of the disclosure, DNAs encoding partialor full-length light and heavy chains are inserted into expressionvectors such that the DNA molecules are operatively linked totranscriptional and translational control sequences. In this context,the term “operatively linked” means that an antibody gene is ligatedinto a vector such that transcriptional and translational controlsequences within the vector serve their intended function of regulatingthe transcription and translation of the DNA molecule. The expressionvector and expression control sequences are chosen to be compatible withthe expression host cell used. The antibody light chain gene and theantibody heavy chain gene can be inserted into separate vectors, or bothgenes can be inserted into the same expression vector. The antibodygenes are inserted into the expression vector by any suitable methods(e.g., ligation of complementary restriction sites on the antibody genefragment and vector, or homologous recombination-based DNA ligation).The light and heavy chain variable regions of the antibodies describedherein can be used to create full-length antibody genes of any antibodyisotype and subclass by inserting them into expression vectors alreadyencoding heavy chain constant and light chain constant regions of thedesired isotype and subclass such that the V_(H) segment is operativelylinked to the C_(H) segment(s) within the vector and the V_(L) segmentis operatively linked to the C_(L) segment within the vector.Additionally or alternatively, the recombinant expression vector canencode a signal peptide that facilitates secretion of the antibody chainfrom a host cell. The antibody chain gene can be cloned into the vectorsuch that the signal peptide is linked in-frame to the amino terminus ofthe antibody chain gene. The signal peptide can be an immunoglobulinsignal peptide or a heterologous signal peptide (i.e., a signal peptidefrom a non-immunoglobulin protein).

In addition to the antibody sequences, the expression vectors of thedisclosure typically carry regulatory sequences that control theexpression of the antibody sequences in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel (GeneExpression Technology. Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990)). It will be appreciated by those skilled in theart that the design of the expression vector, including the selection ofregulatory sequences, may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Examples of regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., theadenovirus major late promoter (AdMLP) and polyoma. Alternatively,nonviral regulatory sequences may be used, such as the ubiquitinpromoter or β-globin promoter. Still further, regulatory elementscomposed of sequences from different sources, such as the SR promotersystem, which contains sequences from the SV40 early promoter and thelong terminal repeat of human T cell leukemia virus type 1 (Takebe, Y.et al. (1988) Mol. Cell. Biol. 8:466-472).

In addition to the antibody chain genes and regulatory sequences, theexpression vectors may carry additional sequences, such as sequencesthat regulate replication of the vector in host cells (e.g., origins ofreplication) and selectable marker genes. The selectable marker genefacilitates selection of host cells into which the vector has beenintroduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and5,179,017, all by Axel et al.). For example, typically the selectablemarker gene confers resistance to drugs, such as G418, hygromycin ormethotrexate, on a host cell into which the vector has been introduced.Selectable marker genes include the dihydrofolate reductase (DHFR) gene(for use in dhfr-host cells with methotrexate selection/amplification)and the neo gene (for G418 selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell byany suitable techniques. The various forms of the term “transfection”are intended to encompass a wide variety of techniques commonly used forthe introduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is possible toexpress the antibodies of the disclosure in either prokaryotic oreukaryotic host cells, expression of antibodies in eukaryotic cells, andtypically mammalian host cells, is most typical.

The present disclosure further provides a host cell containing a nucleicacid molecule provided by the present disclosure. The host cell can bevirtually any cell for which expression vectors are available. It maybe, for example, a higher eukaryotic host cell, such as a mammaliancell, a lower eukaryotic host cell, such as a yeast cell, and may be aprokaryotic cell, such as a bacterial cell. Methods of introducing arecombinant nucleic acid into a host cell are known in the art,including, for example, by calcium phosphate transfection, DEAE, dextranmediated transfection, electroporation or phage infection.

Suitable prokaryotic hosts for transformation include E. coli, Bacillussubtilis, Salmonella typhimurium and various species within the generaPseudomonas, Streptomyces, and Staphylococcus.

Suitable eukaryotic hosts for transformation include yeast, insect(e.g., S2 cells), and mammalian cells. Mammalian host cells forexpressing a binding molecule of the disclosure include, for example,Chinese Hamster Ovary (CHO) cells (including dhfr-CHO cells, describedin Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220 (1980);Sharp, J. Mol. Biol. 159:601-621 (1982)), NSO myeloma cells, COS cells,HEK293F cells, HEK293Tcells, and Sp2 cells. In particular, for use withNSO myeloma or CHO cells, another expression system is the GS (glutaminesynthetase) gene expression system disclosed in WO 87/04462, WO 89/01036and EP 338,841. In some embodiments, antibodies of the presentdisclosure are produced in CHO cells. In some embodiments, antibodies ofthe present disclosure are modified, and do not include a C-terminallysine residue (e.g., the C-terminal lysine residue of an antibody heavychain described herein is removed (such as before or during antibodyproduction)). When expression vectors encoding antibody genes areintroduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using any suitableprotein purification methods known in the art (e.g., protein Achromatography and/or ion exchange chromatography).

VI. Compositions

In other aspects, the present disclosure provides a compositioncontaining a binding molecule (e.g., an antibody or activatableantibody) provided by the disclosure. In one aspect, the composition isa pharmaceutical composition comprising a binding molecule (e.g., anantibody or activatable antibody) and a pharmaceutically acceptablecarrier. The compositions can be prepared by conventional methods knownin the art.

In some embodiments, present disclosure provides a compositioncomprising a binding molecule (e.g., an antibody or activatableantibody) provided by the present disclosure and a pharmaceuticallyacceptable carrier, wherein said binding molecule comprises a variabledomain comprising the HVR amino acid sequence disclosed herein, andwherein said composition comprises not more than about 11%, 10%, 8%, 5%,3%, or 2% of said binding molecule (e.g., an antibody or activatableantibody) that is glycosylated at the asparagine of said amino acidsequence compared with the total amount of binding molecule (e.g., anantibody or activatable antibody) present in said composition. Inanother embodiment, the composition comprises at least about 2% of saidbinding molecule (e.g., an antibody or activatable antibody) that isglycosylated at the asparagine of said amino acid sequence compared withthe total amount of binding molecule (e.g., an antibody or activatableantibody) present in said composition.

The term “pharmaceutically acceptable carrier” refers to any inactivesubstance that is suitable for use in a formulation for the delivery ofa binding molecule. A carrier may be an anti-adherent, binder, coating,disintegrant, filler or diluent, preservative (such as antioxidant,antibacterial, or antifungal agent), sweetener, absorption delayingagent, wetting agent, emulsifying agent, buffer, and the like. Examplesof suitable pharmaceutically acceptable carriers include water, ethanol,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like) dextrose, vegetable oils (such as olive oil), saline, buffer,buffered saline, and isotonic agents such as sugars, polyalcohols,sorbitol, and sodium chloride.

The compositions may be in any suitable forms, such as liquid,semi-solid, and solid dosage forms. Examples of liquid dosage formsinclude solution (e.g., injectable and infusible solutions),microemulsion, liposome, dispersion, or suspension. Examples of soliddosage forms include tablet, pill, capsule, microcapsule, and powder. Aparticular form of the composition suitable for delivering a bindingmolecule (e.g., an antibody or activatable antibody) is a sterileliquid, such as a solution, suspension, or dispersion, for injection orinfusion. Sterile solutions can be prepared by incorporating theantibody in the required amount in an appropriate carrier, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the binding molecule (e.g., an antibody or activatableantibody) into a sterile vehicle that contains a basic dispersion mediumand other carriers. In the case of sterile powders for the preparationof sterile liquid, methods of preparation include vacuum drying andfreeze-drying (lyophilization) to yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof. The various dosage forms of thecompositions can be prepared by conventional techniques known in theart.

The relative amount of a binding molecule (e.g., an antibody oractivatable antibody) included in the composition will vary dependingupon a number of factors, such as the specific binding molecule andcarriers used, dosage form, and desired release and pharmacodynamiccharacteristics. The amount of a binding molecule (e.g., an antibody oractivatable antibody) in a single dosage form will generally be thatamount which produces a therapeutic effect, but may also be a lesseramount. Generally, this amount will range from about 0.01 percent toabout 99 percent, from about 0.1 percent to about 70 percent, or fromabout 1 percent to about 30 percent relative to the total weight of thedosage form.

In addition to the binding molecule (e.g., an antibody or activatableantibody), one or more additional therapeutic agents may be included inthe composition. Examples of additional therapeutic agents are describedherein below. The suitable amount of the additional therapeutic agent tobe included in the composition can be readily selected by a personskilled in the art, and will vary depending on a number of factors, suchas the particular agent and carriers used, dosage form, and desiredrelease and pharmacodynamic characteristics. The amount of theadditional therapeutic agent included in a single dosage form willgenerally be that amount of the agent which produces a therapeuticeffect, but may be a lesser amount as well.

Any of the binding molecules (e.g., antibodies or activatableantibodies) and/or compositions (e.g., pharmaceutical compositions)described herein may be used in the preparation of a medicament (e.g., amedicament for use in treating or delaying progression of cancer in asubject in need thereof).

VII. Use of the Binding Molecules and Pharmaceutical Compositions

Binding molecules (e.g., antibodies or activatable antibodies) andpharmaceutical compositions provided by the present disclosure areuseful for therapeutic, diagnostic, or other purposes, such asmodulating an immune response, treating cancer, enhancing efficacy ofother cancer therapy, enhancing vaccine efficacy, or treating autoimmunediseases. Thus, in other aspects, the present disclosure providesmethods of using the binding molecules (e.g., antibodies or activatableantibodies) or pharmaceutical compositions. In one aspect, the presentdisclosure provides a method of treating a disorder in a mammal, whichcomprises administering to the mammal in need of treatment an effectiveamount of a binding molecule (e.g., an antibody or activatable antibody)or composition provided by the present disclosure. The binding molecule(e.g., an antibody or activatable antibody) may be a CTLA4 antibody(e.g., a human anti-human CTLA4 antibody) or a CTLA4 activatableantibody. In some embodiments, the mammal is a human.

In some embodiments, the disorder is a cancer. A variety of cancers maybe treated or prevented with a method, use, composition, or medicamentprovided by the present disclosure. Examples of such cancers includelung cancers such as bronchogenic carcinoma (e.g., squamous cellcarcinoma, small cell carcinoma, large cell carcinoma, andadenocarcinoma), alveolar cell carcinoma, bronchial adenoma,chondromatous hamartoma (noncancerous), and sarcoma (cancerous); heartcancer such as myxoma, fibromas, and rhabdomyomas; bone cancers such asosteochondromas, condromas, chondroblastomas, chondromyxoid fibromas,osteoid osteomas, giant cell tumors, chondrosarcoma, multiple myeloma,osteosarcoma, fibrosarcomas, malignant fibrous histiocytomas, Ewing'stumor (Ewing's sarcoma), and reticulum cell sarcoma; brain cancer suchas gliomas (e.g., glioblastoma multiforme), anaplastic astrocytomas,astrocytomas, oligodendrogliomas, medulloblastomas, chordoma,Schwannomas, ependymomas, meningiomas, pituitary adenoma, pinealoma,osteomas, hemangioblastomas, craniopharyngiomas, chordomas, germinomas,teratomas, dermoid cysts, and angiomas; cancers in digestive system suchas leiomyoma, epidermoid carcinoma, adenocarcinoma, leiomyosarcoma,stomach adenocarcinomas, intestinal lipomas, intestinal neurofibromas,intestinal fibromas, polyps in large intestine, and colorectal cancers;liver cancers such as hepatocellular adenomas, hemangioma,hepatocellular carcinoma, fibrolamellar carcinoma, cholangiocarcinoma,hepatoblastoma, and angiosarcoma; kidney cancers such as kidneyadenocarcinoma, renal cell carcinoma, hypernephroma, and transitionalcell carcinoma of the renal pelvis; bladder cancers; hematologicalcancers such as acute lymphocytic (lymphoblastic) leukemia, acutemyeloid (myelocytic, myelogenous, myeloblastic, myelomonocytic)leukemia, chronic lymphocytic leukemia (e.g., Sezary syndrome and hairycell leukemia), chronic myelocytic (myeloid, myelogenous, granulocytic)leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B cell lymphoma,mycosis fungoides, and myeloproliferative disorders (includingmyeloproliferative disorders such as polycythemia vera, myelofibrosis,thrombocythemia, and chronic myelocytic leukemia); skin cancers such asbasal cell carcinoma, squamous cell carcinoma, melanoma, Kaposi'ssarcoma, and Paget's disease; head and neck cancers; eye-related cancerssuch as retinoblastoma and intraoccular melanocarcinoma; malereproductive system cancers such as benign prostatic hyperplasia,prostate cancer, and testicular cancers (e.g., seminoma, teratoma,embryonal carcinoma, and choriocarcinoma); breast cancer; femalereproductive system cancers such as uterine cancer (endometrialcarcinoma), cervical cancer (cervical carcinoma), cancer of the ovaries(ovarian carcinoma), vulvar carcinoma, vaginal carcinoma, fallopian tubecancer, and hydatidiform mole; thyroid cancer (including papillary,follicular, anaplastic, or medullary cancer); pheochromocytomas (adrenalgland); noncancerous growths of the parathyroid glands; pancreaticcancers; and hematological cancers such as leukemias, myelomas,non-Hodgkin's lymphomas, and Hodgkin's lymphomas.

In another aspect, the present disclosure provides a method of enhancingan immune response in a mammal, which comprises administering to themammal an effective amount of a binding molecule (e.g., an antibody oractivatable antibody) or composition provided by the present disclosure.In some embodiments, the binding molecule is a CTLA4 antibody orantigen-binding fragment thereof and the mammal is a human. In someembodiments, the binding molecule is a CTLA4 activatable antibody andthe mammal is a human. The term “enhancing immune response” or itsgrammatical variations, means stimulating, evoking, increasing,improving, or augmenting any response of a mammal's immune system. Theimmune response may be a cellular response (i.e. cell-mediated, such ascytotoxic T lymphocyte mediated) or a humoral response (i.e. antibodymediated), and may be a primary or secondary immune response. Examplesof enhancement of immune response include activation of PBMCs and/or Tcells (including increasing secretion of one or more cytokines such asIL-2 and/or IFNγ). The enhancement of immune response can be assessedusing a number of in vitro or in vivo measurements known to thoseskilled in the art, including, but not limited to, cytotoxic Tlymphocyte assays, release of cytokines, regression of tumors, survivalof tumor bearing animals, antibody production, immune cellproliferation, expression of cell surface markers, and cytotoxicity.Typically, methods of the present disclosure enhance the immune responseby a mammal when compared to the immune response by an untreated mammalor a mammal not treated using the recited methods.

In practicing the therapeutic methods, the binding molecules (e.g.,antibodies or activatable antibodies) may be administered alone asmonotherapy, or administered in combination with one or more additionaltherapeutic agents or therapies. Thus, in another aspect, the presentdisclosure provides a combination therapy, which comprises a bindingmolecule (e.g., an antibody or activatable antibody) in combination withone or more additional therapies or therapeutic agents for separate,sequential or simultaneous administration. The term “additionaltherapeutic agent” may refer to any therapeutic agent other than abinding molecule (e.g., an antibody or activatable antibody) provided bythe disclosure. In one particular aspect, the present disclosureprovides a combination therapy for treating cancer in a mammal, whichcomprises administering to the mammal an effective amount of a bindingmolecule (e.g., an antibody or activatable antibody) provided herein incombination with one or more additional therapeutic agents. In a furtherembodiment, the mammal is a human.

A wide variety of cancer therapeutic agents may be used in combinationwith a binding molecule (e.g., an antibody or activatable antibody)provided by the present disclosure. One of ordinary skill in the artwill recognize the presence and development of other cancer therapieswhich can be used in combination with the methods and binding moleculese.g., antibodies or activatable antibodies) of the present disclosure,and will not be restricted to those forms of therapy set forth herein.Examples of categories of additional therapeutic agents that may be usedin the combination therapy for treating cancer include (1)chemotherapeutic agents, (2) immunotherapeutic agents, and (3) hormonetherapeutic agents. In some embodiments, the additional therapeutic is aviral gene therapy, an immune checkpoint inhibitor, a target therapy, aradiation therapies, vaccination therapies, and/or a chemotherapeutic.

The term “chemotherapeutic agent” refers to a chemical or biologicalsubstance that can cause death of cancer cells, or interfere withgrowth, division, repair, and/or function of cancer cells. Examples ofchemotherapeutic agents include those that are disclosed in WO2006/129163, and US 20060153808, the disclosures of which areincorporated herein by reference. Examples of particularchemotherapeutic agents include: (1) alkylating agents, such aschlorambucil (LEUKERAN), mcyclophosphamide (CYTOXAN), ifosfamide (IFEX),mechlorethamine hydrochloride (MUSTARGEN), thiotepa (THIOPLEX),streptozotocin (ZANOSAR), carmustine (BICNU, GLIADEL WAFER), lomustine(CEENU), and dacarbazine (DTIC-DOME); (2) alkaloids or plant vincaalkaloids, including cytotoxic antibiotics, such as doxorubicin(ADRIAMYCIN), epirubicin (ELLENCE, PHARMORUBICIN), daunorubicin(CERUBIDINE, DAUNOXOME), nemorubicin, idarubicin (IDAMYCIN PFS,ZAVEDOS), mitoxantrone (DHAD, NOVANTRONE). dactinomycin (actinomycin D,COSMEGEN), plicamycin (MITHRACIN), mitomycin (MUTAMYCIN), and bleomycin(BLENOXANE), vinorelbine tartrate (NAVELBINE)), vinblastine (VELBAN),vincristine (ONCOVIN), and vindesine (ELDISINE); (3) antimetabolites,such as capecitabine (XELODA), cytarabine (CYTOSAR-U), fludarabine(FLUDARA), gemcitabine (GEMZAR), hydroxyurea (HYDRA), methotrexate(FOLEX, MEXATE, TREXALL), nelarabine (ARRANON), trimetrexate(NEUTREXIN), and pemetrexed (ALIMTA); (4) Pyrimidine antagonists, suchas 5-fluorouracil (5-FU); capecitabine (XELODA), raltitrexed (TOMUDEX),tegafur-uracil (UFTORAL), and gemcitabine (GEMZAR); (5) taxanes, such asdocetaxel (TAXOTERE), paclitaxel (TAXOL); (6) platinum drugs, such ascisplatin (PLATINOL) and carboplatin (PARAPLATIN), and oxaliplatin(ELOXATIN); (7) topoisomerase inhibitors, such as irinotecan(CAMPTOSAR), topotecan (HYCAMTIN), etoposide (ETOPOPHOS, VEPESSID,TOPOSAR), and teniposide (VUMON); (8) epipodophyllotoxins(podophyllotoxin derivatives), such as etoposide (ETOPOPHOS, VEPESSID,TOPOSAR); (9) folic acid derivatives, such as leucovorin (WELLCOVORIN);(10) nitrosoureas, such as carmustine (BiCNU), lomustine (CeeNU); (11)inhibitors of receptor tyrosine kinase, including epidermal growthfactor receptor (EGFR), vascular endothelial growth factor (VEGF),insulin receptor, insulin-like growth factor receptor (IGFR), hepatocytegrowth factor receptor (HGFR), and platelet-derived growth factorreceptor (PDGFR), such as gefitinib (IRESSA), erlotinib (TARCEVA),bortezomib (VELCADE), imatinib mesylate (GLEEVEC), genefitinib,lapatinib, sorafenib, thalidomide, sunitinib (SUTENT), axitinib,rituximab (RITUXAN, MABTHERA), trastuzumab (HERCEPTIN), cetuximab(ERBITUX), bevacizumab (AVASTIN), and ranibizumab (LUCENTIS), lym-1(ONCOLYM), antibodies to insulin-like growth factor-1 receptor (IGF-1R)that are disclosed in WO2002/053596); (12) angiogenesis inhibitors, suchas bevacizumab (AVASTIN), suramin (GERMANIN), angiostatin, SU5416,thalidomide, and matrix metalloproteinase inhibitors (such as batimastatand marimastat), and those that are disclosed in WO2002055106; and (13)proteasome inhibitors, such as bortezomib (VELCADE).

The term “immunotherapeutic agents” refers to a chemical or biologicalsubstance that can enhance an immune response of a mammal. Examples ofimmunotherapeutic agents include: bacillus Calmette-Guerin (BCG);cytokines such as interferons; vaccines such as MyVax personalizedimmunotherapy, Onyvax-P, Oncophage, GRNVAC1, Favld, Provenge, GVAX,Lovaxin C, BiovaxID, GMXX, and NeuVax; and antibodies such asalemtuzumab (CAMPATH), bevacizumab (AVASTIN), cetuximab (ERBITUX),gemtuzunab ozogamicin (MYLOTARG), ibritumomab tiuxetan (ZEVALIN),panitumumab (VECTIBIX), rituximab (RITUXAN, MABTHERA), trastuzumab(HERCEPTIN), tositumomab (BEXXAR), ipilimumab (YERVOY) tremelimumab,CAT-3888, agonist antibodies to OX40 receptor (such as those disclosedin WO2009/079335), agonist antibodies to CD40 receptor (such as thosedisclosed in WO2003/040170, and TLR-9 agonists (such as those disclosedin WO2003/015711, WO2004/016805, and WO2009/022215).

The term “hormone therapeutic agent” refers to a chemical or biologicalsubstance that inhibits or eliminates the production of a hormone, orinhibits or counteracts the effect of a hormone on the growth and/orsurvival of cancerous cells. Examples of such agents suitable for themethods herein include those that are disclosed in US20070117809.Examples of particular hormone therapeutic agents include tamoxifen(NOLVADEX), toremifene (Fareston), fulvestrant (FASLODEX), anastrozole(ARIMIDEX), exemestane (AROMASIN), letrozole (FEMARA), megestrol acetate(MEGACE), goserelin (ZOLADEX), and leuprolide (LUPRON). The bindingmolecules of this disclosure may also be used in combination withnon-drug hormone therapies such as (1) surgical methods that remove allor part of the organs or glands which participate in the production ofthe hormone, such as the ovaries, the testicles, the adrenal gland, andthe pituitary gland, and (2) radiation treatment, in which the organs orglands of the patient are subjected to radiation in an amount sufficientto inhibit or eliminate the production of the targeted hormone.

In some embodiments, the additional therapeutic agent is one or more ofpomalyst, revlimid, lenalidomide, pomalidomide, thalidomide, aDNA-alkylating platinum-containing derivative, cisplatin,5-fluorouracil, cyclophosphamide, an anti-CD137 antibody, an anti-PD-1antibody, an anti-PD-L1 antibody, an anti-CD20 antibody, an anti-CD40antibody, an anti-DR5 antibody, an anti-CD1d antibody, an anti-TIM3antibody, an anti-SLAMF7 antibody, an anti-KIR receptor antibody, ananti-OX40 antibody, an anti-HER2 antibody, an anti-ErbB-2 antibody, ananti-EGFR antibody, cetuximab, rituximab, trastuzumab, pembrolizumab,radiotherapy, single dose radiation, fractionated radiation, focalradiation, whole organ radiation, IL-12, IFNα, GM-CSF, a chimericantigen receptor, adoptively transferred T cells, an anti-cancervaccine, and an oncolytic virus.

The combination therapy for treating cancer also encompasses thecombination of a binding molecule (e.g., an antibody or activatableantibody) with surgery to remove a tumor. The binding molecule (e.g., anantibody or activatable antibody) may be administered to the mammalbefore, during, or after the surgery.

The combination therapy for treating cancer also encompasses combinationof a binding molecule (e.g., an antibody or activatable antibody) withradiation therapy, such as ionizing (electromagnetic) radiotherapy(e.g., X-rays or gamma rays) and particle beam radiation therapy (e.g.,high linear energy radiation). The source of radiation can be externalor internal to the mammal. The binding molecule (e.g., an antibody oractivatable antibody) may be administered to the mammal before, during,or after the radiation therapy.

The binding molecules (e.g., antibodies or activatable antibodies) andcompositions provided by the present disclosure can be administered viaany suitable enteral route or parenteral route of administration. Theterm “enteral route” of administration refers to the administration viaany part of the gastrointestinal tract. Examples of enteral routesinclude oral, mucosal, buccal, and rectal route, or intragastric route.“Parenteral route” of administration refers to a route of administrationother than enteral route. Examples of parenteral routes ofadministration include intravenous, intramuscular, intradermal,intraperitoneal, intratumor, intravesical, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, transtracheal,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal, subcutaneous, or topical administration. The bindingmolecules (e.g., antibodies or activatable antibodies) and compositionsof the present disclosure can be administered using any suitable method,such as by oral ingestion, nasogastric tube, gastrostomy tube,injection, infusion, implantable infusion pump, and osmotic pump. Thesuitable route and method of administration may vary depending on anumber of factors such as the specific binding molecule (e.g., anantibody or activatable antibody) being used, the rate of absorptiondesired, specific formulation or dosage form used, type or severity ofthe disorder being treated, the specific site of action, and conditionsof the patient, and can be readily selected by a person skilled in theart.

The term “effective amount” of a binding molecule (e.g., an antibody oractivatable antibody) may refer to an amount that is effective for anintended therapeutic purpose. For example, in the context of enhancingan immune response, an “effective amount” may be any amount that iseffective in stimulating, evoking, increasing, improving, or augmentingany response of a mammal's immune system. In the context of treating adisease, an “effective amount” may be any amount that is sufficient tocause any desirable or beneficial effect in the mammal being treated.Specifically, in the treatment of cancer, examples of desirable orbeneficial effects include inhibition of further growth or spread ofcancer cells, death of cancer cells, inhibition of reoccurrence ofcancer, reduction of pain associated with the cancer, or improvedsurvival of the mammal. The therapeutically effective amount of abinding molecule (e.g., an antibody or activatable antibody) usuallyranges from about 0.001 to about 500 mg/kg, and more usually about 0.01to about 100 mg/kg, of the body weight of the mammal. For example, theamount can be about 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, 50mg/kg, or 100 mg/kg of body weight of the mammal. In some embodiments,the therapeutically effective amount of a binding molecule (e.g., anantibody or activatable antibody) is in the range of about 0.01-30 mg/kgof body weight of the mammal. In some other embodiments, thetherapeutically effective amount of a binding molecule (e.g., anantibody or activatable antibody) is in the range of about 0.05-15 mg/kgof body weight of the mammal. The precise dosage level to beadministered can be readily determined by a person skilled in the artand will depend on a number of factors, such as the type, and severityof the disorder to be treated, the particular binding molecule (e.g., anantibody or activatable antibody) employed, the route of administration,the time of administration, the duration of the treatment, theparticular additional therapy employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A binding molecule (e.g., an antibody or activatable antibody) orcomposition is usually administered on multiple occasions. Intervalsbetween single doses can be, for example, daily, weekly, monthly, everythree months or yearly. An exemplary treatment regimen entailsadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every three months oronce every three to six months. Typical dosage regimens for a bindingmolecule (e.g., an antibody or activatable antibody) include 1 mg/kgbody weight or 3 mg/kg body weight via intravenous administration, usingone of the following dosing schedules: (i) every four weeks for sixdosages, then every three months; (ii) every three weeks; (iii) 3 mg/kgbody weight once followed by 1 mg/kg body weight every three weeks.

VIII. Kits

In another aspect, provided herein is a kit comprising a bindingmolecule (e.g., an antibody or activatable antibody) and/or compositionof the present disclosure. In some embodiments, the kit furthercomprises a package insert comprising instructions for use of thebinding molecule (e.g., an antibody or activatable antibody) and/orcomposition. In some embodiments, the kit further comprises one or morebuffers, e.g., for storing, transferring, administering, or otherwiseusing the binding molecule (e.g., an antibody or activatable antibody)and/or composition. In some embodiments, the kit further comprises oneor more containers for storing the binding molecule (e.g., an antibodyor activatable antibody) and/or composition.

The foregoing written description is considered to be sufficient toenable one skilled in the art to practice the present disclosure. Thefollowing Examples are offered for illustrative purposes only, and arenot intended to limit the scope of the present disclosure in any way.Indeed, various modifications of the present disclosure in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and fall within the scope ofthe appended claims.

EXAMPLES Example 1: Generation of Primary Fabs that Specifically Bind toHuman CTLA4

Proprietary phagemid libraries (see PCT application numberPCT/CN2017/098333, incorporated herein by reference in its entirety; seealso PCT application number PCT/CN2017/098299, incorporated herein byreference in its entirety) were employed to pan against human CTLA4antigens. A total of 3-5 rounds of panning were conducted. After thefinal round of panning, single-colony supernatant ELISA was performed toidentify the primary hits that specifically recognized human CTLA4 (seee.g., UniProt Accession Number P16410). The primary hits were defined asthose whose ELISA signals were at least twice that of background. Thehits were then sequenced, and the unique clones were expressed in E.coli and purified. Their affinities against human CTLA4 were measured byForteBio Octet RED96 Systems. Briefly, AHC sensors (Anti-Human IgG FcCapture Dip and Read Biosensors) were used to capture recombinant humanCTLA4-Fc (Sino Biological, 11159-H03H), and dipped into wells containingpurified Fabs that were diluted to 10 μg/mL in kinetic buffer (0.02%Tween20, 0.1% BSA in PBS buffer). The acquired ForteBio data wereprocessed with Data Acquisition software 7.1, and kinetic data werefitted to a 1:1 Langmuir binding model. The list of candidates wasrefined to 234 Fab hits with both ELISA positive hits and uniquesequences. Following the criteria of K_(D) response signal R>0.1,R²>0.9, the list was further refined to 43 hits of interest. Theaffinity and kinetic parameters (with background subtracted) of thesehits are shown in Table 1 below.

TABLE 1 affinities of selected Fabs for human CTLA4 Hit ID: K_(D) (M):kon (1/Ms): koff (1/s): B18153 5.96E−09 8.93E+04 5.32E−04 B157196.93E−09 3.21E+05 2.22E−03 B15746 7.15E−09 3.07E+05 2.20E−03 B157347.63E−09 3.05E+05 2.33E−03 B15710 8.01E−09 3.51E+05 2.81E−03 B138741.06E−08 1.19E+05 1.26E−03 B13898 1.07E−08 1.09E+05 1.16E−03 B157281.18E−08 4.24E+05 4.99E−03 B15705 1.20E−08 1.94E+05 2.33E−03 B157161.21E−08 3.91E+04 4.71E−04 B15672 1.23E−08 6.83E+04 8.39E−04 B157381.24E−08 3.76E+05 4.67E−03 B15694 1.28E−08 1.52E+05 1.94E−03 B157541.35E−08 2.76E+05 3.71E−03 B15749 1.38E−08 2.69E+05 3.72E−03 B157401.45E−08 1.78E+05 2.58E−03 B15489 1.46E−08 6.08E+04 8.87E−04 B138801.59E−08 1.04E+05 1.66E−03 B15699 1.66E−08 4.42E+05 7.35E−03 B157451.73E−08 2.58E+05 4.47E−03 B18174 1.82E−08 5.16E+04 9.38E−04 B157391.86E−08 4.24E+05 7.86E−03 B15721 2.21E−08 5.31E+05 1.17E−02 B156732.44E−08 6.95E+04 1.70E−03 B15737 2.75E−08 4.77E+05 1.31E−02 B156962.79E−08 1.44E+05 4.03E−03 B15491 2.98E−08 6.24E+04 1.86E−03 B157243.01E−08 5.52E+05 1.66E−02 B15741 3.08E−08 2.09E+05 6.44E−03 B157573.72E−08 1.05E+05 3.91E−03 B15759 5.36E−08 2.46E+05 1.32E−02 B157565.55E−08 1.20E+05 6.63E−03 B15722 6.12E−08 7.09E+04 4.34E−03 B157506.23E−08 9.37E+04 5.84E−03 B15702 6.92E−08 3.61E+05 2.50E−02 B142427.58E−08 3.36E+05 2.55E−02 B13878 8.59E−08 3.08E+04 2.64E−03 B157171.05E−07 1.77E+05 1.85E−02 B15187 1.37E−07 2.73E+04 3.75E−03 B156951.52E−07 1.81E+05 2.76E−02 B15189 1.70E−07 2.04E+04 3.47E−03 B157621.85E−07 4.60E+04 8.49E−03 B15730 2.59E−07 3.45E+04 8.93E−03

Next, the species cross-reactivity of various Fab hits was determined byELISA. Briefly, 100 μL of 1.25 μg/mL anti-human IgG (Fab specific)antibody (Sigma, 15260) was coated on a Maxisorp microplate (ThermoScientific 446469) at 4° C. overnight. After blocking, 100 μL of the Fabhits (2 μg/mL) were added and incubated for one hour. After washing thewells 3-4 times, serial dilutions of human, cynomolgus monkey, or mouseCTLA4 antigens fused with human FC fragments were added and incubatedfor one hour. After washing, HRP-labelled goat anti-human FC was diluted1:2000 with PBS, and added to each well for a one hour incubation.Plates were washed three times and incubated with TMB substrate for 3-5minutes at room temperature. Absorbance at 450 nm was measured after thereaction was stopped. Species cross-reactivity for each of the testedFabs is summarized in Table 2 below. Interestingly, this analysisidentified Fabs with varying cross-reactivities: the results indicatedthat hits B13873, B15700, B15704, B15706, B15709, B15711, B15712,B15715, B15720, B15725, B15723, B15731, B15732, B15735, B15736, B15744,B15760, B16083, and B15188 bound to human, monkey and mouse CTLA4; hitsB15188, B15190, B15701, B15729, B15733, B15742, B15747, B15743, B15751,B15752, B15753 and B18157 bound to human and monkey CTLA4, and weaklybind to mouse CTLA4; hits B13878, B14242, B15189, B15491, B15673,B15694, B15696, B15699, B15702, B15705, B15710, B15716, B15717, B15719,B15721, B15722, B15724, B15728, B15734, B15737, B15738, B15739, B15740,B15745, B15746, B15749, B15750, B15754, B15756, B15757, B15759 andB15762 bound to human CTLA4, but not to mouse CTLA4; hit B15688 bound tohuman and mouse CTLA4, but not to monkey CTLA4; and hits B13874, B13880,B13898, B15187, B15489, B15672, B15695, B15730, B15741, B18153 andB18174 bound to human CTLA4, but not to monkey or mouse CTLA4.

TABLE 2 Fab cross-reactivities with human, monkey, and mouse CTLA4 BindsBinds Binds Hit ID: Human CTLA? Monkey CTLA4? Mouse CTLA4? B13873 YesYes Yes B15688 Yes No Yes B15700 Yes Yes Yes B15704 Yes Yes Yes B15706Yes Yes Yes B15709 Yes Yes Yes B15711 Yes Yes Yes B15712 Yes Yes YesB15715 Yes Yes Yes B15720 Yes Yes Yes B15723 Yes Yes Yes B15725 Yes YesYes B15731 Yes Yes Yes B15732 Yes Yes Yes B15735 Yes Yes Yes B15736 YesYes Yes B15744 Yes Yes Yes B15760 Yes Yes Yes B16083 Yes Yes Yes B15188Yes Yes weak B15190 Yes Yes weak B15701 Yes Yes weak B15729 Yes Yes weakB15733 Yes Yes weak B15742 Yes Yes weak B15743 Yes Yes weak B15747 YesYes weak B15751 Yes Yes weak B15752 Yes Yes weak B15753 Yes Yes weakB18157 Yes Yes weak B13874 Yes No No B13878 Yes Yes No B13880 Yes No NoB13898 Yes No No B14242 Yes Yes No B15187 Yes No No B15189 Yes Yes NoB15489 Yes No No B15491 Yes Yes No B15672 Yes No No B15673 Yes Yes NoB15694 Yes Yes No B15695 Yes No No B15696 Yes Yes No B15699 Yes Yes NoB15702 Yes Yes No B15705 Yes Yes No B15710 Yes Yes No B15716 Yes Yes NoB15717 Yes Yes No B15719 Yes Yes No B15721 Yes Yes No B15722 Yes Yes NoB15724 Yes Yes No B15728 Yes Yes No B15730 Yes No No B15734 Yes Yes NoB15737 Yes Yes No B15738 Yes Yes No B15739 Yes Yes No B15740 Yes Yes NoB15741 Yes No No B15745 Yes Yes No B15746 Yes Yes No B15749 Yes Yes NoB15750 Yes Yes No B15754 Yes Yes No B15756 Yes Yes No B15757 Yes Yes NoB15759 Yes Yes No B15762 Yes Yes No B18153 Yes No No B18174 Yes No No

Example 2: IgG Conversion and Expression

13 of the refined hits from Example 1 above were then converted intohuman IgG1 antibodies for detailed biophysical and functionalcharacterization (Table 3). The heavy and light chains of Fab HitsB15709, B15716, B15722, B15732, B15740, B15744, B15756, B15700, B15711,B15717, B15735, B15736 and B16083 were separately cloned into themammalian expression vector pTT5-SPB. The heavy and light chain of areference antibody was also cloned into pTT5-SPB.

TABLE 3 Fab hits cloned as IgG1 antibodies Antibody (AB) Name: Fab HitID: Isotype: TY21585 B15709 hIgG1 TY21586 B15716 hIgG1 TY21587 B15722hIgG1 TY21588 B15732 hIgG1 TY21589 B15740 hIgG1 TY21580 B15744 hIgG1TY21591 B15756 hIgG1 TY21687 B15700 hIgG1 TY21688 B15711 hIgG1 TY21689B15717 hIgG1 TY21680 B15735 hIgG1 TY21691 B15736 hIgG1 TY21692 B16083hIgG1 TAC2114 Reference hIgG1

Pairs of plasmids encoding the antibody heavy and lights chains weretransiently transfected into 293F cells following the manufacturer'sprotocol. The supernatants of cells transfected with plasmids encodingantibodies TY21585, TY21586, TY21587, TY21588, TY21589, TY21580, orTY21591 were harvested, cleared by centrifugation and filtration, andthe resulting IgGs were purified with standard protein A affinitychromatography (MabSelect SuRe, GE Healthcare). The proteins were elutedand neutralized, and buffer exchanged into 20 mM PB buffer (20 mMNaH2PO4, 150 mM NaCl, pH 7.0). Protein concentrations were determined byUV-spectrophotometry, and IgG purity was analyzed under denaturing,reducing and non-reducing conditions by SDS-PAGE or SEC-HPLC.

The supernatants of cells transfected with plasmids encoding antibodiesTY21687, TY21689, TY21680, TY21691, or TY21692 were harvested, clearedby centrifugation and filtration, and the resulting IgGs were purifiedwith standard protein A affinity chromatography (MabSelect SuRe, GEHealthcare). The proteins were eluted and neutralized, and bufferexchanged into 20 mM Histidine buffer (20 mM Histidine, 3.5 mL 6 M HCl,pH5.5). Protein concentrations were determined by UV-spectrophotometry,and IgG purity was analyzed under denaturing, reducing and non-reducingconditions by SDS-PAGE or SEC-HPLC.

Example 3: In Vitro Functional Characterization of IgG-ConvertedAntibodies

Binding affinity and kinetics of antibodies against human, monkey andmouse CTLA4 were examined (Table 4) by surface plasmon resonance (SPR)analysis using a Biacore™ T200 instrument (Biacore AB, Uppsala, Sweden)according to the manufacturer's guidelines. Anti-Human IgG (Fc) antibodyfrom Human Antibody Capture Kit (GE BR-1008-39) was immobilized on CM5chips by coupling of its amine groups onto carboxylated surfaces ofsensor chips according to the instructions of an Amine Coupling kit (GEBiacore #BR-1000-50). The immobilized Anti-Human IgG (Fc) antibody wasused to capture antibodies TY21585, TY21586, TY21580, TY21591, TY21687,TY21689, TY21680, TY21691, TY21692 and TAC2114. TAC2114 has the sameamino acid sequence as the commercial antibody Ipilimumab. Binding wasmeasured at six different concentrations (3.13, 6.25, 12.5, 25, 50, and100 nM diluted in running buffer), and a flow rate of 30 μI/min wasused. The running buffer used was HBS-EP (100 mM HEPES, 1.5M sodiumchloride, 0.05% sur-factant P20, pH 7.6). The association anddissociation curves were fitted to a 1:1 Langmuir binding model usingBiacore T200 Evaluation Software (Biacore AB) according to themanufacturer's guidelines. As shown in Table 4 below, all of the testedantibodies were able to bind human and monkey CTLA4, and all antibodiesexcept for TY21591, TY21689, and TAC2114 were also capable of binding tomouse CTLA4.

TABLE 4 binding affinity of IgG1 antibodies to human, monkey, and mouseCTLA4 K_(D) (nM) Ab name: Human CTLA4: Monkey CTLA4: Mouse CTLA4:TY21585 8.15 8.26 1.71 TY21586 7.80 15.00 303 TY21580 2.58 0.44 0.51TY21591 9.68 3.27 NC TY21687 1.82 1.52 2.76 TY21689 0.95 0.91 NC TY216801.48 1.29 0.67 TY21691 2.08 2.44 1.46 TY21692 3.95 2.76 1.12 TAC21146.68 1.98 NC NC = not cross-reactive.

The ability of certain IgG antibodies to bind to soluble human (FIG. 1A,Table 5A) or canine (FIG. 1B, Table 5B) CTLA4 was next tested. 1 μg/mLhuman CTLA4 fused with human FC fragment, or canine CTLA4 fused with Histag, were prepared and used to coat an ELISA plate at 2-8° C. overnight.After blocking, 100 μL serial diluted IgG antibodies were added andincubated at 37° C. for 1 hour. Plates were washed for four times andthen incubated with HRP-anti human IgG (Fab specific) (1:6000 dilution)at 37° C. for 1 hour. Plates were washed again four times and incubatedwith TMB substrate for 15 minutes at room temperature. Absorbance at 450nm was measured after the reaction was stopped. The data was analyzed byGraphpad Prism 6 with nonlinear fitting. As shown in FIGS. 1A-B andTables 5A-B, all tested antibodies bound to human CTLA4, and, with theexception of antibodies TY21586 and TAC2114, also bound to canine CTLA4.Interestingly, TY21580 bound both human and dog CTLA4 with the highestaffinity, with K_(D)s of 0.27 and 0.49, respectively.

TABLE 5A ELISA for human CTLA4 EC₅₀: K_(D) Ab name: LogEC₅₀: M nM nMTY21585 −9.256 5.552E−10 0.5552 0.56 TY21586 −8.896  1.27E−09 1.27101.27 TY21580 −9.571 2.685E−10 0.2685 0.27 TY21591 −9.080 8.309E−100.8309 0.83 TY21687 −9.495 3.201E−10 0.3201 0.32 TAC2114 −9.4823.296E−10 0.3296 0.33

TABLE 5B ELISA for canine CTLA4 EC₅₀: K_(D) Ab name: LogEC₅₀: M nM nMTY21585 −8.219 6.045E−09 6.0450 6.05 TY21586 ND ND ND ND TY21580 −9.313 4.86E−10 0.4860 0.49 TY21591 −9.151 7.066E−10 0.7066 0.71 TY21687−9.128 7.451E−10 0.7451 0.75 TAC2114 ND ND ND ND ND = not determined.

The affinities of the antibodies were also assessed against human andmouse CTLA4 transiently expressed on the surface of HEK293F cells (FIG.2). Briefly, HEK293F cells were transfected with a plasmid expressingfull-length human, monkey or mouse CTLA4 from a bicistronic IRES vectoralso encoding EGFP, and EGFP expression was used to identify thetransfected cells. After 48 hours, the mammalian cell suspension(2×10⁵/well) was transferred to an Eppendorf tube, centrifuged, thesupernatant was discarded, the cells were resuspended with 1 mL PBSA (toa density is 4×10⁶ cells/mL), and added to a 96 well plate. 3-foldserial dilutions of the test antibodies (15 μg/mL, 5 μg/mL, 1.67 μg/mL,0.55 μg/mL, 0.185 μg/mL, 0.062 μg/mL, and 0.0309 μg/mL, plus a 0 μg/mLblank control) were pipetted into the 96-well plate, incubated on icefor 1 hour (protected from light), the cells were washed withpre-chilled 1×PBSA buffer, and subsequently incubated with an AlexaFluor® 647 conjugated mouse anti-human FC antibody for 30 minutes onice. The cells were then washed once prior to analysis by flow cytometry(Beckman® CytoFlex). As shown in FIG. 2, all test antibodies werecapable of binding human CTLA4 expressed on the surface of HEK293Fcells, and, with the exception of TY21589, all antibodies bound to mousecell-surface exposed mouse CTLA4. TY21580 bound to human and mouse CTLA4expressed on the cell surface with low nM affinity, whereas antibodiesTY21585 and TY21586 bound to human CTLA4 expressed on the cell surfacewith high nM affinities.

Binding affinity and kinetics of antibodies TY21580, TY21687, TY21680and TY21691 against rat CTLA4 protein were also tested using a ForteBiored 96 instrument (Pall, USA). SA sensors (Pall, 185019) were used toimmobilize biotinylated rat CTLA4 protein fused with human FC, and thesensors were then contacted with the IgG-converted hits at aconcentration of 15 μg/mL (diluted with KB Buffer, PBS buffersupplemented with 0.02% Tween 20 and 0.1% BSA) for 300 seconds, thendissociated in KB buffer for 300 seconds. The association anddissociation curves were fitted to a 1:1 Langmuir binding model usingForteBio Data Analysis 7.1 (Pall, USA) according to the manufacturer'sguidelines. As shown in Table 6 below, all of the tested antibodies wereable to bind to rat CTLA4.

TABLE 6 binding affinity of test antibodies for rat CTLA4 Ab name: K_(D)(nM): TY21580 0.38 TY21687 0.78 TY21680 0.21 TY21691 0.58Binding of IgGs to Activated T Cells

Next, the ability of the IgGs to bind to activated human, monkey, andmouse T cells was tested. Human PBMCs were freshly isolated from theblood of a healthy donor (#106) by density gradient centrifugation usingHistopaque-1077 (Sigma). Human T cells were isolated from PBMCs usingthe Human T Cell Enrichment Kit (StemCell Technologies), followed bystimulation with anti-CD3 and anti-CD28 antibodies. Briefly, anti-CD3antibody (Clone: OKT3, BioLegend) was coated at 0.2 μg in 200 μL perwell in a 96 well plate at 4° C. overnight. After washing, T cellssuspended in RPMI-1640 containing 10% FBS and 1% Penn/Strep were addedto the plate. 5×10E5 T cells in 200 μL were added to each well of the 96well plate. Then 1 μL of anti-human CD28 antibody (Clone: 28.2, BD) wasadded to a final concentration of 5 μg/mL. T cells were incubated for 96hours, and then the binding of TY21580 to T cells was determined by flowcytometric analysis (FIG. 3). T cells were stained with APC-labeledTY21580 or human IgG1 (isotype control) for 2 hours at 37° C. Afterwashing, cells were analyzed on the CytoFLEX flow cytometer (BeckmanCoulter) and the data was analyzed with FlowJo software. As shown inFIG. 3, TY21580 bound to activated CD4+ and CD8+ human T cells, whilecontrol IgG showed no binding. In addition, APC-TY21580 showed nobinding to resting T cells (data not shown).

Monkey PBMCs were freshly isolated from the blood of a naïve cynomolgusmonkey by density gradient centrifugation using Histopaque-1077 (Sigma).Monkey T cells were isolated from PBMCs using the Pan T cell Isolate KitNon-human primate (Miltenyi Biotec), followed by stimulation withanti-CD3 and anti-CD28 antibodies. Briefly, anti-CD3 antibody (Clone:SP34, BD) was coated at 0.2 μg in 200 μL per well in a 96 well plate at4° C. overnight. After washing, T cells suspended in RPMI-1640containing 10% FBS and 1% Penn/Strep were added to the plate. 2×10E5 Tcells in 200 μL were added to each well of the 96 well plate. Then 1 μLof anti-human CD28 antibody (Clone: 28.2, BD) was added to a finalconcentration of 5 μg/mL. T cells were incubated for 72 h, and bindingof TY21580 to the T cells was determined by flow cytometric analysis(FIG. 3). T cells were stained with APC-labeled TY21580 or human IgG1(isotype control) for 2 h at 37° C. After washing, cells were analyzedon the CytoFLEX flow cytometer (Beckman Coulter) and the data wasanalyzed with FlowJo software. As shown in FIG. 3, TY21580 bound toactivated CD4+ and CD8+ monkey T cells, while control IgG showed nobinding. In addition, APC-TY21580 showed no binding to resting T cells(data not shown).

Mouse T cells isolated from adult BALB/c mouse spleens were used toinduce CTLA-4 expression. Splenocytes from fresh mouse spleens were usedto isolate T cells with the EasySep™ Mouse T Cell Isolation kit(StemCell Technologies), followed by stimulation with anti-mouse CD3 andanti-CD28 antibodies. Briefly, anti-mouse CD3ε antibody (Biolegend) wascoated at 0.2 μg in 200 μL per well in a 96 well plate at 4° C.overnight. After washing, mouse T cells suspended in RPMI-1640containing 10% FBS and 1% Penn/Strep were added to each well of theplate at 5×10E5 T cells in 200 μL. Then 1 μL of anti-mouse CD28 antibody(eBioscience) was added to a final concentration of 5 μg/mL. Mouse Tcells were incubated for 72 h and then the binding of TY21580 to T cellswas determined by flow cytometric analysis (FIG. 3). T cells werestained with APC-labeled TY21580 or human IgG1 (isotype control) for 2 hat 37° C. After washing, cells were analyzed on the CytoFLEX flowcytometer (Beckman Coulter) and the data was analyzed with FlowJosoftware. As shown in FIG. 3, TY21580 bound to activated mouse CD4+ andCD8+ T cells, while control IgG showed no binding.

Binding Selectivity of Antibodies for Human CTLA4

Antibody selectivity was next examined. Human CTLA4, PD1, LAG3, Tim3,B7H3, CD95, TNFR1, OX40, CD40, PD-L1, BLTA, VISTA, PDL2, ICOS and B7H4were transiently overexpressed on the surface of HEK293F cells.Transfected cells were washed in pre-chilled 1×PBSA buffer (1.76 mMKH2PO4, 10.14 mM Na2HPO4.12H2O, 2.68 mM KCl, 136.89 mM NaCl and 1% BSA),then incubated with 100 nM test antibodies for 1 hour on ice. Cells werewashed once with staining buffer, and Alexa Fluor® 647 conjugated mouseanti-human FC antibodies were added and incubated for 30 minutes on ice,protected from light. Samples were washed once with staining bufferprior to analysis by flow cytometry. TY21585 TY21586, TY21580, TY21687,TY21689, TY21680 and TY21691 were tested in human CTLA4, PD1, LAG3,Tim3, and B7-H3 (FIG. 4A); TY21585 TY21586, TY21580 were further testedin human CD95, TNFR1, OX40, and CD40 (FIG. 4B); in addition, TY21586,TY21580 were tested in human PD-L1, BLTA, VISTA, PDL2, ICOS and B7-H4(FIG. 4C). As shown in FIGS. 4A-C, all tested antibodies bound speciallyto human CTLA4, and not to any other tested antigen (or parental cellstransfected with empty vectors).

Ligand Competition Binding by ELISA

Antibodies were then tested for their ability to block binding of CTLA4to its cognate ligands CD80 and CD86 by ELISA. Recombinant human CTLA4(fused with human Fc and His tag) was diluted to 1 μg/mL in carbonatebuffer solution, pH 9.4, and coated on a Maxisorp plate at 4° C.overnight. Plates were blocked with PBS supplemented with 2% (w/v) skimmilk at 37° C. for 1 hour. After washing, 50 uL biotinylated CD80 (4μg/mL) and 50 uL of various concentrations of test antibodies (2-foldserial dilutions ranging from 200 μg/mL to 1.56 g/mL) were addedsuccessively to each well and incubated at 37° C. for 1 hour. Plateswere washed four times, and 100 μL HRP-neutravidin (1:1000) was added toeach well and incubated at 37° C. for 1 hour. Plates were washed aspreviously described, and 50 μL TMB substrate solution was added andincubated at room temperature for 5 minutes before the reaction wasstopped by 50 μL sulfate acid (2M). As shown in FIGS. 5A-B, all testedantibodies blocked binding of CTLA4 to CD80, except for TY21589.

Recombinant human CD86 fused with human Fc was diluted to 1 μg/mL incarbonate buffer solution, pH 9.4, and coated on a Maxisorp plate at 4°C. overnight. Plates were blocked with PBS supplemented with 2% (w/v)skim milk at 37° C. for 1 hour. After washing, 50 uL biotinylated humanCTLA4 fused with human FC and His tag (2.8 μg/mL), and 50 uL of variousconcentrations of test antibodies (2-fold serial dilutions ranging from100 μg/mL to 0.78 μg/mL) were added successively to each well andincubated at 37° C. for 1 hour. Plates were washed four times, and 100μL HRP-neutravidin (1:1000) were added to each well and incubated at 37°C. for 1 hr. Plates were washed as previously described, and 50 μL TMBsubstrate solution was added and incubated at room temperature for 5minutes before the reaction was stopped by 50 μL sulfate acid (2M). Asshown in FIGS. 5C-D, all tested antibodies blocked binding of CTLA4 toCD86.

Ligand Competition Binding by Flow Cytometry

Antibodies were also tested for their ability to block binding of CTLA4to its cognate ligands CD80 and CD86 by flow cytometry. The plasmidencoding full-length human CTLA4 was transiently expressed in HEK293Fcells. Cells were washed with staining buffer (PBSA buffer including1.76 mM KH₂PO₄, 10.14 mM Na₂HPO₄.12H₂O, 2.68 mM KCl, 136.89 mM NaCl and1% BSA), and resuspended in staining buffer containing 100 nM testantibodies. After incubation on ice for 60 minutes, 100 nM biotinylatedhuman CD80-Fc-Bio or CD86-Fc-Bio was added to each well and incubatedfor another 1 hour on ice. Cells were washed with staining buffer once,and 100 μL staining buffer containing Alexa fluor 633 conjugatedstreptavidin were added and incubated on ice for 30 minutes, protectedfrom light. Cells were washed once, and analyzed by CytoFlex flowcytometry. As shown in FIG. 6A, all tested antibodies blocked binding ofCTLA4 to CD80 in a concentration dependent manner. TY21588 showed thestrongest blocking capability, followed by TY21580 and TAC2114 withsignificant blocking; and TY21585, TY21587, TY21589, TY21591 with lesseffective blocking. TY21589 showed little to no blocking. As shown inFIG. 6B, all tested antibodies blocked binding of CTLA4 to CD86 in aconcentration dependent manner. TY21588, TY21589, TY21580, TY21591 andTAC2114 showed the strongest blocking capability; TY21585 and TY21587had less effective blocking.

Binding to FcγR

Binding affinity of TY21586, TY21580 and TAC2114 against CD16a (176Phe)(Sino Biological Inc, 10389-H08H), CD16a (176Val, 10389-H08H1), CD32a(Sino Biological Inc, 10374-H08H), CD32b (Sino Biological Inc,10259-H08H), and CD64 (Sino Biological Inc, 10256-H08H) was next tested.Protein binding was examined by surface plasmon resonance (SPR) analysisusing a Biacore™ T200 instrument (Biacore AB, Uppsala, Sweden) accordingto the manufacturer's guidelines. Protein L (Sino Biological Inc.11044-H07E) was immobilized on CM5 chips by coupling of its amine groupsonto carboxylated surfaces of sensor chips according to the instructionsof an Amine Coupling kit (GE Biacore #BR-1000-50). The immobilizedAnti-Human IgG (Fc) antibody was used to capture TY21586, TY21580 andTAC2114. Serial concentrations (12.5, 25, 50, 25, 100 and 200 nM)diluted in running buffer of FcγR protein were injected at a flow rateof 30 μI/min. The running buffer used was HBS-EP (100 mM HEPES, 1.5Msodium chloride, 0.05% sur-factant P20, pH 7.6). The association anddissociation curves were fitted to a 1:1 Langmuir binding model usingBiacore T200 Evaluation Software (Biacore AB, Uppsala, Sweden) accordingto the manufacturer's guidelines. As shown in Table 7 below, TY21586 andTY21580 showed similar affinity for binding to FcγR, as compared to thereference antibody (TAC2114).

TABLE 7 antibody binding to FcγR K_(D) (nM) CD16a CD16a Ab name (176Phe)(176Val) CD32a CD32b CD64 TY21586 143.000 453.000 884.000 1340.000 0.214TY21580 145.000 624.000 884.000 1110.000 0.202 TAC2114 237.000 577.000706.000 735.000 0.255Binding to FcRn

Binding affinity of test antibodies to recombinant human FcRn wasexamined by surface plasmon resonance (SPR) analysis using a Biacore™T200 instrument (Biacore AB, Uppsala, Sweden) according to themanufacturer's guidelines. Human FcRn protein (Sino Biological Inc.11044-H07E) was immobilized on CM5 chips by coupling of its amine groupsonto carboxylated surfaces of sensor chips according to the instructionsof an Amine Coupling kit (GE Biacore #BR-1000-50). 100 nM of eachantibody was diluted in running buffer (50 mM NaPO4, 150 mM NaCl, and0.05% (v/v) Surfactant 20, pH 6.0), and the samples were injected at aflow rate of 30 μI/min for 120 seconds. As show in FIG. 7, antibodiesTY21585, TY21580, TY21591, TY21687, and TY21691 exhibited higher % boundthan TAC2114 to FcRn, which indicated that the IgG-FcRn complex on theBiacore chip could undergo conformation change that stabilizes thecomplex, as compared with the reference antibody (TAC2114). AntibodiesTY21586, TY21587, TY21589, TY21689 and TY21680 showed low % bound.

Human PBMC Activation

Preliminary studies showed TY21580 did not stimulate human T cellactivation or proliferation. Since CTLA4 activity on T cells is relatedto the first signal (TCR/CD3) and second signals involvingB7-CD28/CTLA-4, human PBMCs were chosen, and the activity of TY21580 inthe presence of low concentration of anti-CD3 was determined. Anti-CD3antibody (OKT-3) was coated on a 96 well plate overnight at 4° C. Afterwashing, 1×10⁵ freshly isolated human PBMCs were added to each well,followed by adding the test articles at different concentrations.Induction of IL-2 was measured 48 hours after stimulation using a HumanIL-2 ELISA Ready-SET-Go (Invitrogen) kit. IFNγ in the supernatant wasmeasured using a Human IFNγ ELISA Ready-SET-Go (Invitrogen) kit. Asshown in FIG. 8A and FIG. 9, antibody TY21580 significantly increasedhuman PBMC activation in the presence of anti-CD3, while TY21580 alonehad no activity.

Dendritic Cell MLR Assay

DC-MLR assays were conducted using monocyte derived DCs and CD4+ Tlymphocytes in three donor pairs: D42/D109, D32/D104, and D104/D42 (FIG.10). To get DC cells, PBMCs were isolated by density gradientcentrifugation from a healthy donor, and CD14+ monocytes were purifiedfrom PBMCs using a positive selection commercial kit (StemCell). CD14+monocytes were skewed into DCs by in vitro culturing in RPMI-1640supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin,20 ng/mL rhGM-CSF and 20 ng/mL rhIL-4 for 6 days. Culture medium waschanged with fresh medium on day 3. DC maturation was induced inRPMI-1640 medium supplemented with 10% heat-inactivated FBS, 1%penicillin/streptomycin, and 50 ng/mL rhTNF-α on day 6 for 24 hours.CD4+ T cells were purified by negative isolation from another healthydonor. Test articles were titrated into corresponding concentrations (asshown in FIG. 10). Collected DCs (1×10⁴) were co-cultured with allogenicCD4+ T cells (1×10⁵) with or without titrated test articles. Anti-PD1antibody was used as positive control for DC-MLR assays. On day 5 afterco-culture, IFNγ was measured in the supernatant by ELISA using a humanIFNγ Ready-SET-Go ELISA kit. As shown in FIG. 10, antibody TY21580showed weak activity in the DC-MLR assay using human CD4+ T cells andDCs.

ADCC Activity of Antibody TY21580

HEK293F cells overexpressing human CTLA-4 were used as target cells toevaluate TY21580-mediated ADCC activity. Human NK cells were freshlyisolated from human PBMCs using a human NK isolation kit (StemCell).1×10⁵ NK cells and 1×10⁴ HEK293F/hCTLA-4 cells (E:T ratio 10:1) weremixed with different concentrations of antibody. After incubation for 4hours, LDH was measured to determine the ADCC activity. The % lysis wasthen calculated using the following formula: % Lysis=[(ExperimentalRelease)−Ave (Target+NK)]/[Ave (Target Max)−Ave (Target only)]×100%. Asshown in FIGS. 11A-B, TY21580 showed stronger ADCC activity than areference antibody (TAC2114). Isotype control showed no ADCC activitywhatsoever.

ADCC activity was also evaluated using human Treg cells (A, donor #96;B, donor #12) and NK cells (A, donor #99; .B, donor #05). To get humanTreg cells, human PBMCs were freshly isolated from a healthy donor, andTreg cells were negatively selected using an EASYSEP™ Human Regulatory TCell Enrichment Kit (StemCell Technologies). Enriched human Treg cellswere further expanded by CD3/CD28 stimulation in the presence of IL-2,and confirmed by CD25 and FOXP3 staining and FACS analysis. To get humanNK cells, human PBMCs were freshly isolated from another healthy donor,and NK cells were isolated using a Human NK isolation kit (StemCellTechnologies). Human Treg cells were labeled with 10 μM Calcein-AM(Invitrogen) at 37° C. for 30 min. After washing three times, labeledTreg cells were mixed with different concentration of test articles,followed by the addition of NK cells. 1×10⁵ NK cells and 1×10⁴ oflabeled human Treg cells were added to the wells of a 96 well plate, andmixed to make the E:T ratio 10:1. After 4 hours of incubation,calcein-AM concentration in the supernatant was measured to determinethe ADCC activity using the following formula: % Lysis=[(ExperimentalRelease)−Ave (Target+NK)]/[Ave (Target Max)−Ave (Target only)]×100%. Asshown in FIGS. 12A-B, antibody TY21580 showed stronger ADCC activitythan the reference antibody (TAC2114). Isotype control showed no ADCCactivity.

CDC Activity of TY21580

HEK293F cells overexpressing human CTLA-4 were labeled with 10 μMCalcein-AM (Invitrogen) at 37° C. for 30 min. To the wells of the 96well plate, antibodies of different concentrations were mixed with 1×10⁴labeled cells and 5% normal human serum complement (NHSC, Quidel). After5 hours of incubation, calcein-AM in the supernatant was measured todetermine the CDC activity (FIG. 13).

Human PBMCs were freshly isolated from a healthy donor (donor #57). CD4+T cells were isolated using an EasySep Human CD4+ T cell enrichment KIT(StemCell), and stimulated with PMA (50 ng/mL)+Ionmycin (1 μM) for 20hours to induce CTLA-4 expression on the cell surface. Activated humanCD4+ T cells were then labeled with 10 μM Calcein-AM (Invitrogen) at 37°C. for 30 minutes. To the wells of the 96 well plate, antibodies ofdifferent concentrations were mixed with 1×10⁴ labeled human CD4+ Tcells and 5% normal human serum complement (NHSC, Quidel). After 5 hoursof incubation, calcein-AM in the supernatant was measured to determinethe CDC activity (FIG. 14). TY21580 showed no CDC activity againstHEK293F/hCTLA-4 cells or activated human T cells.

Taken together, these results indicate that the antibodies describedherein were capable of binding to human CTLA4 with high affinity andspecificity, and such antibodies efficiently blocked the interaction ofCTLA4 with its cognate ligands CD86 and CD80. The antibodies were alsoshown to be cross-reactive with CTLA4 from multiple species.Furthermore, binding to CTLA4 could modulate T cell activation andinduce ADCC activity against CTLA4-expressing cell such as Tregs.

Example 4: In Vivo Characterization of IgG-Converted Antibodies

As described in the Examples above, the species cross-reactivity (humanand mouse) of the antibodies allowed for the determination of theanti-tumor potency of the antibodies in multiple syngeneic tumor models,including MC38 and CT26 colorectal tumor models, an H22 liver tumormodel, a PAN02 pancreatic tumor model, and a 3LL lung tumor model.

Anti-Tumor Efficacy in an MC38 Colorectal Tumor Model

C57BL/6 mice (n=8 per group, female, 6-8 weeks old) were inoculatedsubcutaneously with MC38 (NTCC-MC38) murine colon cancer cells. Whentumors were established (80 mm³), treatment began with isotype controlantibody and three different dosages of antibody TY21580 byintraperitoneal injection, twice a week for three weeks. Tumor growthwas monitored twice a week and reported as the mean tumor volume±s.e.m.over time (FIGS. 15A-C). As shown in FIG. 15A, compared to the isotypecontrol antibody, TY21580 exhibited potent in vivo anti-tumor activitywith tumors completely regressing at all three dosages. As shown in FIG.15B, up to sixty days post-treatment, 8 out of 8 mice in the 10 mg/kg ofTY21580 group, 7/8 in the 2.5 mg/kg of TY21580 group, 6/8 in the 0.5mg/kg of TY21580 group, remained tumor free. The long lasting memory ofimmunity against MC38 tumor cells was demonstrated when the mice in the10 mg/kg of TY21580 group were re-challenged, as shown in FIG. 15C.

Anti-Tumor Efficacy in a CT26 Colorectal Tumor Model

BALB/c mice (n=8 per group, female, 7-8 weeks old) were inoculatedsubcutaneously with CT26 (Shanghai Institutes for Biological Sciences)murine colon cancer cells. When tumors were established (70 mm³),treatment began with isotype control antibody and two different dosagesof antibody TY21580 by intraperitoneal injection, twice a week. Tumorgrowth was monitored twice a week and reported as the mean tumorvolume±s.e.m. over time. As shown in FIG. 16, compared to the isotypecontrol antibody, TY21580 exhibited potent in vivo anti-tumor activitywith almost 100% inhibition at dosages as low as 0.1-1 mg/kg.

Anti-Tumor Efficacy in an H22 Liver Tumor Model

BALB/c mice (n=5 per group, female, 7-8 weeks old) were inoculatedsubcutaneously with H22 (China Center for Type Culture Collection)murine liver cancer cells. When tumors were established (60 mm³),treatment began with isotype control antibody, antibody TY21586 at threedifferent dosages (0.1 mg/kg, 1 mg/kg, 5 mg/kg), or antibody TY21580 attwo different dosages (0.1 mg/kg, 1 mg/kg) by intraperitoneal injection,twice a week. Tumor growth was monitored twice a week and reported asthe mean tumor volume±s.e.m. over time. As shown in FIG. 17, compared tothe isotype control antibody, both TY21580 and TY21586 exhibited potentin vivo anti-tumor activity in a dose-dependent manner. When compared atthe same dosage, TY21580 was more potent than TY21586 in this tumormodel. In addition, TY21580 administration at 1 mg/kg led to tumorregression.

Anti-Tumor Efficacy in a Lewis Lung Tumor Model

C57BL/6 mice (n=6 per group, female, 8 weeks old) were inoculatedsubcutaneously with Lewis (JenNio Bio, Guandong, China) murine lungcancer cells. When tumors were established (70 mm³), treatment beganwith isotype control antibody, or antibodies TY21580, TY21687, TY21680,or TY21691, all at a dosage of 5 mg/kg by intraperitoneal injection,twice a week. Tumor growth was monitored twice a week and reported asthe mean tumor volume±s.e.m. over time. As shown in FIG. 18, compared tothe isotype control antibody, antibodies TY21580, TY21687, and TY21680showed significant inhibition of tumor growth, while antibody TY21691did not show potent anti-tumor activity.

Anti-Tumor Efficacy in a PAN02 Pancreatic Tumor Model

C57BL/6 mice (n=8 per group, female, 6 weeks old) were inoculatedsubcutaneously with PAN-02 (CAMS Cell Culture Center) murine pancreaticcancer cells. When tumors were established (85 mm³), treatment beganwith isotype control antibody, or antibody TY21580 at three differentdosages (0.5 mg/kg, 2 mg/kg, 0.5 mg/kg), by intraperitoneal injection,twice a week. Tumor growth was monitored twice a week and reported asthe mean tumor volume±s.e.m. over time. As shown in FIG. 19, compared tothe isotype control antibody, TY21580 showed potent anti-tumor activityin a dose-dependent manner.

Anti-Tumor Efficacy of Antibody TY21580 Monotherapy, or in Combinationwith Anti-CD137 Antibody in a 3LL Lung Tumor Model

C57BL/6 mice (n=10 per group, female, 6-8 weeks old) were inoculatedsubcutaneously with 3LL (JCRB) murine lung cancer cells. When tumorswere established (75 mm³), treatment began with isotype controlantibody, TY21580 (10 mg/kg), anti-CD137 (10 mg/kg), or the combinationof TY21580 and anti-CD137 by intraperitoneal injection, twice a week.Anti-CD137 is a proprietary monoclonal antibody developed that possessesthe ability to bind both human and murine CD137 (see PCT applicationnumber PCT/CN2017/098332, incorporated herein by reference in itsentirety). Tumor growth was monitored twice a week and reported as themean tumor volume±s.e.m. over time. As shown in FIGS. 20A-B, compared tothe isotype control antibody, both TY21580 and anti-CD137 showed potentanti-tumor activity, and the combination inhibited tumor growth morethan either monotherapy alone.

Re-Challenge of Mice with Complete Response to TY21580

BALB/c mice (n=8 per group, female, 7-8 weeks old) were inoculatedsubcutaneously with H22 (China Center for Type Culture Collection)murine liver cancer cells. When tumors were established (60 mm³),treatment began with isotype control antibody, or antibody TY21580 attwo different dosages (1 mg/kg, 10 mg/kg), by intraperitoneal injectiontwice a week for three weeks. Tumor growth was monitored twice a weekand reported as the mean tumor volume±s.e.m. over time. Compared to theisotype control antibody, TY21580 at both dosages lead to complete tumorregression a few days after the last dose, and the mice remained tumorfree 60 days post treatment. Mice in the 10 mg/kg of TY21580 treatmentgroup were then re-challenged on Day 60 subcutaneously with H22 tumorcells in the opposite flank, and monitored for tumor growth. As shown inFIG. 21, these mice remained tumor free after re-challenge with the sametumor cells, suggesting that specific anti-tumor memory was developed inthese mice. A re-challenge control group was set up at the same timewith naïve mice inoculated with the same number of H22 tumor cells, andtheir tumors grew out rapidly.

Antibody Pharmacokinetics

A pharmacokinetic study of antibodies TY21585, TY21586, TY21580 andTY21591 was conducted in BALB/c female mice (at about eight weeks ofage). Three mice per group were intravenously injected with the testantibodies at 10 mg/kg by tail vein injection. Blood samples (˜20 μL persample) were collected at 1 hour, 8 hours, 48 hours, 168 hours, 336hours, and 500 hours post dosing. Blank control blood was collected fromthree naive female mice without antibody administration. Serumconcentrations of each test antibody were determined by ELISA, in whichCTLA4-His-Fc was used for capture, and HRP-labeled anti-human IgG (Fabspecific) antibody (Sigma) was used for detection. As shown in FIG. 22,TY21586 exhibited comparable pharmacokinetics to TAC2114 in mice, whileTY21585, TY21580, and TY21591 were cleared much faster.

A pharmacokinetic study of TY21586 and TY21580 was also conducted innaive cynomolgus monkeys. Each antibody was administered by intravenousbolus injection at 10 mg/kg to one female and one male monkey. Serumsamples were collected pre-dose (0 h) and 0.25 hours, 1 hour, 8 hours,24 hours, 72 hours, 120 hours, 168 hours, 240 hours, 336 hours, 504hours, and 672 hours post dosing. Serum concentrations of TY21586 andTY21580 were determined by ELISA, in which CTLA4-His-Fc was used forcapture, and HRP-labeled anti-human IgG (Fab specific) antibody (Sigma)was used for detection. As shown in FIG. 23 and FIG. 24, compared toTY21586, TY21580 was cleared much more quickly in monkeys, potentiallydue to the rapid increase in anti-drug antibodies observed in theseanimals.

Repeated Dosing Toxicity Studies

Repeated dosing toxicity of TY21580 was conducted in normal BALB/c mice.Vehicle control or antibody TY21580 (at 25 mg/kg or 50 mg/kg) wasadministered i.p. (10 mL/kg) on Day 1, Day 4, Day 7, and Day 11. Fivefemale mice and five male mice (five weeks old) were included in eachgroup. Mice were monitored daily for abnormal behaviors and symptoms,and measured daily for food intake and body weight. On day 14, animalswere euthanized for post-mortem examination, and other analysis. Bloodwas collected from each animal, with up to six blood samples collectedper group (three male, three female) used for hematology (RBC, platelet,WBC, WBC differential) and/or blood biochemistry (ALT, AST, GLB, ALP,and LDH etc.) analysis. The following organs from each mouse werecollected and weighed: heart, lung, thymus, liver, spleen, kidney,testes, and ovaries. The liver samples from 6 animals (three male, threefemale) per group were fixed in FFPE. FFPE blocks for liver tissues wereprepared, sectioned and H&E stained for histopathology analysis.

During the in-life period of the whole study, there was no abnormalbehavior observed, or un-scheduled animal death. Compared to the vehicletreatment, TY21580 did not affect the food intake and body weight of theanimals. Post-mortem examination also did not show any obvious lesionsin mice of the treatment groups at both dosage levels, except that thespleen weight was increased in the TY21580 treated groups (FIG. 25A-B).Hematology analysis did not show any significant changes, as indicatedby the blood biochemistry parameters tested in mice treated withTY21580. No obvious abnormalities were found in the histopathologysections of the liver from the mice (FIG. 26). Overall, TY21580 was welltolerated in this study, with no significant toxicity observed in mice.

Taken together, these results indicate that the CTLA4 antibodiesdescribed herein were very safe to mice, had potent anti-tumor activity,and could induce long-lasting immune memory against tumor cells.

Example 5: Antibody Developability Profile

For developability assessment, purified TY21586 and TY21580 wereexchanged into storage buffer (20 mM histidine, pH 5.5). Allexperiments, including solubility, stability under accelerated stressconditions, and differential scanning fluorescence (DSF) tests wereperformed in storage buffer. For all SEC-HPLC analyses, TSKgel columns(Tosoh Bioscience G3000SWxl) were used.

Antibody Solubility

Samples containing antibodies TY21586 or TY21580 were formulated at aconcentration greater than 100 mg/mL in storage buffer, and the amountof high molecular weight (HMW) protein aggregates was tested (Table 8).Antibodies then were adjusted to about 12 mg/mL in storage buffer.Samples (12 μg each) were then assayed through SEC-HPLC for detection ofhigh molecular weight protein aggregates. As shown in FIG. 27, nosignificant increase of HMW aggregates was observed at antibodiesformulated at high concentrations (above 100 mg/mL) for 30 min.

TABLE 8 antibody solubility Ab name Concentration (mg/mL) Aggregation(HMW %) TY21586 197.8 0 TY21580 126.0 +0.10Antibody Stability Under Accelerated Stress Conditions

Antibody stabilities were also examined under accelerated stressconditions. Results of these experiments are summarized in Table 9 andFIG. 28. TY21586 and TY21580 remained stable after 6 cycles of freezing(−80° C.) and thawing (room temperature). After seven days at 50° C.,there was little change of HMW aggregate or low molecular weight (LMW)fragments. In longer-term time course experiments (40° C. for up to 28days), TY21586 and TY21580 remained stable, and there were nosignificant increases of HMW aggregates or LMW fragments.

TABLE 9 changes of HMX % under accelerated stress conditions Freeze- 50°C. 40° C. thaw 50° C. 7 d 40° C. 28 d Ab name 6 cycles 7 d LMW % 28 dLMW % TY21586 5.34 0 2.14 −0.22 1.37 TY21580 0.49 0 2.02 0.02 0.43

Furthermore, thermostability (as measured by differential scanningfluorescence (DSF)) showed that both TY21586 and TY21580 were stable upto at least about 55° C. The transition midpoint (Tm), thecharacteristic temperature at which the unfolding transition for almostall protein domains occur, is shown Table 10 below.

TABLE 10 thermostability by DSF Ab name Tm onset (° C.) Tm1 (° C.) Tm2(° C.) TY21586-16Z01 55 67.26 76.40 TY21580-16Z01 55 67.64 76.50

Finally, it was found that the highest achievable concentration ofantibodies TY21586 and TY21580 was over 197.8 mg/mL and 126.0 mg/mL,respectively, after centrifugation.

Taken together, these results indicate that even without formulationoptimization, the CTLA4 antibodies TY21586 and TY21580 had excellentdevelopability profile.

Example 6: Methods of Identifying Self-Blocking Peptides forTY21580-Derived CTLA4 Activatable Antibodies

Described herein is a new system that has been designed and executed forefficient discovery of masking moieties with good developability. Inthis system, the target antibody fragments, either Fab (FIG. 29) or scFv(FIG. 30), were first displayed on the yeast surface, and were confirmedto be functional in binding to its antigen. Then the improved peptidelibraries were directly fused to the N-terminus of the light chain of aCTLA4 antibody (TY21580), and a yeast library was constructed thatdisplayed the fusion protein on the yeast surface. The yeast librarythen underwent several rounds of FACS-based screening: first the yeastclones that had low binding to antigen were enriched, then the enrichedyeast clones were treated with a protease to remove the N-terminalpeptide, and the clones with high binding to antigen were selected(FIGS. 29 and 30). After 4-5 rounds of sorting, the plasmids wereextracted from these clones and the masking peptide sequences wereconfirmed through DNA sequencing.

Example 7: Design of Constrained Peptide Libraries (CPLs) for CTLA4Activatable Antibodies

Four exemplary constrained peptide libraries (CPLs) were designed (Table11).

TABLE 11 Designed CPLs CPL name: Amino Acid Sequence: CPL010EVGSY(Z₆)C(Z₆)C(Z₂)SGRSA (SEQ ID NO: 152) CPL011EVGSY(Z₆)C(X₆)C(Z₂)SGRSA (SEQ ID NO: 153) CPL012EVGSY(Z₆)C(Z₈)C(Z₂)SGRSA (SEQ ID NO: 154) CPL013EVGSY(Z₆)C(X₈)C(Z₂)SGRSA (SEQ ID NO: 155) Each X is independently anamino acid selected from the group consisting of A, C, D, E, F, G, H, I,K, L, M, N, P, Q, R, S, T, V, W, and Y; each Z is independently an aminoacid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V,H, and P

At their cores were the sequences Z₆CX₆CZ₂ (SEQ ID NO: 137) or Z6CX8CZ2(SEQ ID NO: 138), and the two fixed cysteine residues formed a disulfidebond to constrain the peptide conformations. In the synthesizedoligonucleotides, the degenerate codon NHC was adopted in all placesexcept inside the loop, where an NNK codon was also employed in CPL011and CPL013. In contrast to the NNK or NNS codon, NHC codon encodes 12residues (Table 12), encompassing significant diversity, but lacking thechemically labile residues methionine, tryptophan, and cysteine. Inaddition, the reduced theoretical diversity compared with the NNK or NNScodon enabled the construction of libraries with better coverage.

TABLE 12 NHC codons NHC: AAC ACC ATC TAC TCC TTC GAC GCC GTC CAC CCC CTCAmino N T I Y S F D A V H P L acid:

Following these masking peptide sequences was an invariant cleavagepeptide sequence (SGRSAGGGGSPLGLAGSGGS, SEQ ID NO: 180) containing twoprotease recognition sites: SGRSA (SEQ ID NO: 149) for the proteaseurokinase-type plasminogen activator (uPA), and PLGLAG (SEQ ID NO: 150)for the proteases matrix metalloproteinase-2 (MMP-2) and matrixmetalloproteinase-9 (MMP-9). These recognition sites have been used bymany group in in vivo tumor cell-specific activation of targeting agents(see e.g., Ke et al. (1997) J Biol Chem 272(33):20456-62; Gerspach etal. (2006) Cancer Immunol Immunother 55(12):1590-600; and Jiang et al.(2004) Proc Natl Acad Sci USA 101(51):17867-72). During yeast-basedscreening, the MMP-9 recognition sequence was replaced with the TobaccoEtch Virus (TEV) protease recognition sequence (ENLYFQG, SEQ ID NO: 151)due to the availability and specificity of the TEV protease.

The CPLs and the invariant cleavage peptide were fused to the N-terminusof light chain of the target antibody (TY21580), in the form of eitherscFv or Fab, that is connected to the yeast surface displayed Aga2protein. The inclusion of the surrogate TEV protease recognition sitewas important in identifying the right type of masking peptidesequences, i.e, the antigen binding is blocked before protease cleavage,and antigen binding is enabled after protease cleavage. The examplesdescribed below demonstrated that the cleavage-activation mechanism ofactivatable antibodies initially shown in yeast was replicated in fullIgG molecules expressed in mammalian cells.

Example 8: Construction and Validation of TY21580-Derived ActivatableAntibodies Targeting CTLA4

Display of the Functional Target Antibody on the Yeast Surface

A low copy number, CEN/ARS-based vector was used to express the targetantibody (antibody TY21580, targeting human CTLA4) under the control ofthe inducible GAL1-10 promoter in the yeast S. cerevisiae. The surfacedisplay of scFvs was achieved through the Aga2 protein fused at itsC-terminus under the control of the GAL1 promoter, similar to previouslypublished arrangements (Boder and Wittrup (1997) Nat Biotechnol15(6):553-7). For Fabs, their surface display was achieved through theAga2 protein fused to the N-terminus of the heavy chain (fusion of VHand CH1), under the control of the GAL1 promoter, while the light chain(fusion of VH and CL) was under the control of the GAL10 promoter. TheFabs were displayed on the yeast surface through its association withthe membrane anchored heavy chain.

The surface display of the Fab or scFv was verified by staining withantibodies recognizing the fused affinity tag, and the functionality ofthe Fabs or scFvs displayed on yeast was examined using biotinylatedhuman CTLA4. Briefly, 48 hours after induction in galactose medium,yeast cells (1×10{circumflex over ( )}6) were harvested, washed oncewith PBSA buffer, and then incubated with 10 nM of biotinylated antigenfor 1 hour at room temperature. The yeast cells were then washed twicewith PBSA buffer, and incubated with PE conjugated streptavidin (1:500dilution) (eBioscience #2-4317-87) for 30 minutes at 4° C. The yeastcells were then analyzed by flow cytometry. As shown in FIGS. 31A-B,both Fabs (FIG. 31A) and scFvs (FIG. 31B) targeting CTLA4 weresuccessfully displayed on the yeast surface, and were both capable ofbinding strongly to their antigens.

Construction of Yeast Libraries Containing CPLs

Synthesized oligonucleotides encoding the CPLs were fused with theoligonucleotides encoding the cleavage peptides through 5 cycles of PCR.The compositions of PCR reactions were: 1× PrimeSTAR buffer, 2.5 mMdNTP, 100 μM of F-primer and R-primer each, and 100 μM each of template1 (CPL oligonucleotide) and template 2 (oligonucleotide encoding thecleavage peptide), and 2.5 U of PrimeSTAR HS DNA Polymerase. The PCRprogram used was: a) 1 cycle of 96° C. for 5 minutes; 2) 5 cycles of 96°C. (15 sec), 60° C. (15 sec), 72° C. (6 sec); and 3) 1 cycle of 72° C.for 3 minutes. Exonuclease I was used to digest the single-stranded DNAbefore purification of the PCR product through gel electrophoresis. Thepurified PCR product was then digested with BamHI and KpnI, and clonedinto a bacterial filter vector digested with the same two restrictionenzymes. In the filter vector, the CPL and the cleavage peptides wereplaced downstream of a bacterial secretion signal peptide, and upstreamof a beta-lactamase lacking signal sequence. The functionalbeta-lactamase, selected on ampicillin plates, indicated in-framefusions of CPLs and the cleavage peptides, thereby eliminating anyout-of-frame errors (N-1 or N-2) introduced into the synthesizeddegenerate oligonucleotides. In addition, some poorly folded sequenceswere also reduced from the pool. The ligation product was transformedinto electro-competent bacterial cells, and the diversity of CPLlibraries was generally between 5×10{circumflex over ( )}9 and1×10{circumflex over ( )}10. Sequencing of individual clones indicatedthat very high in-frame rate (in many cases, almost 100%) were achievedthrough this approach.

To make yeast libraries containing CPLs, the plasmids were extractedfrom the bacterial libraries, and used as templates for PCRamplification of the DNA fragments encoding the CPLs and cleavagepeptide. The amplified PCR fragments were purified throughgel-electrophoresis, and together with a linearized plasmid thatexpressed the target antibody fused to Aga2, were transformed intoelectro-competent yeast cells. The homologous sequences on both ends ofthe PCR fragments and the plasmids ensured efficient homologousrecombination inside yeast cells. The diversity of the constructed yeastlibraries was generally between 1×10{circumflex over ( )}9 to2×10{circumflex over ( )}9.

FACS-Based Screening of Masking Peptides Against a CTLA4 Antibody

A total of 1×10{circumflex over ( )}8 yeast cells from a CPL yeastlibrary were used to screen for masking peptides against the targetantibody. For each round of sorting through MoFlo XDP, yeast cellsinduced in galactose medium were harvested, washed once with PBSAbuffer, and then incubated with 10 nM (decreased to 1 nM in the laterrounds) of biotinylated antigen for 1 hour at room temperature. Theyeast cells were then washed twice with PBSA buffer, and incubated withPE conjugated streptavidin (1:500 dilution) (eBioscience #2-4317-87) for30 minutes at 4° C. After two more washes with PBSA buffer, the yeastcells were adjusted to 2-3 OD/mL, and subject to sorting. As shown inFIG. 32, in round 1, 10 nM of biotinylated CTLA4-Fc was used, and theweak binders were enriched. The yeast cells from round 1, after growthin glucose medium, were induced in galactose medium and treated withAcTEV protease (6U/OD cell) (Thermo Fisher Scientific #12575015) for 2hours at 30° C., and the strong binders were purified. Starting from the3^(rd) round of sorting, the concentration of the biotinylated CTLA4-Fcwas reduced to 1 nM, and the weak binders were collected. At the 4^(th)round, fractions of the yeast cells were also treated with AcTEV inparallel, to verify the protease cleavage mediated activation of thetarget antibody. As shown in FIG. 32, it was apparent that AcTEVcleavage resulted in a dramatic increase of the population of cells thatbound strongly to antigen, suggesting that the screening strategy waseffective. The single clones from the 5^(th) round of sorting wereplated on selective media, and grown individually for furtherconfirmation of cleavage mediated activated antigen binding.

As shown in FIGS. 33A-B, the selected CTLA4 activatable antibody clones,either in scFv (FIG. 33A) or Fab (FIG. 33B) format, exhibited littlebinding to antigen in the presence of masking peptide. However, bindingto antigen was dramatically increased when the yeast cells were treatedwith TEV protease to remove the masking peptide. The incorporation ofthe TEV recognition site in the cleavage peptide, combined with theapplication of TEV protease to verify the selected clones, significantlyincreased the success rate of masking peptide selection.

To identify the masking peptide sequences, the shuttle plasmids wereextracted from the selected yeast clones (Generay #GK2002-200), andtransformed into competent E. coli cells. The plasmids were prepared,and the regions encoding the masking peptides were sequenced andaligned. As anticipated, these sequences could be separated into severalgroups, indicating clear enrichment through rounds of sorting. Fourgroups of masking peptide sequences, together with the invariantcleavage peptide sequences, are listed in Table 13.

TABLE 13 Masking peptide sequences Sample Peptide ID: name: Masking +cleavage peptide sequences: TY22401 B13189EVGSYNFVADSCPDHPYPCSASGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 168) TY22402B13180 EVGSYIVHHSDCDAFYPYCDSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 170)TY22403 B13192 EVGSYYSAYPACDSHYPYCNSSGRSAGGGGSPLGLAGSGGS(SEQ ID NO: 172) TY22404 B13197EVGSYPNPSSDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 174)IgG Conversion and Expression

The four groups of masking peptides listed in Table 13, as well asadditional four masking peptide sequences (Table 14) derived from two ofthem (B13192 and B13197) to eliminate a potential glycosylation site,were converted into IgG1s.

TABLE 14 additional masking peptide sequences Sample ID: Masking +cleavage peptide sequences: TY22563EVGSYYSAYPACDSHYPYCQSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 177) TY22564EVGSYYSAYPACDSHYPYCNSAGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 178) TY22565EVGSYPQPSSDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 179) TY22566EVGSYPNPASDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 180)

The heavy and light chains were cloned into the mammalian expressionvector pCDNA3.3 (Thermo Fisher Scientific, cat #K830001) separately, andthe masking peptides and the invariant cleavage peptide were fused tothe N-terminus of the light chain in the same manner as displayed onyeast surface. The VH and VL sequences for the parental CTLA4 antibody(TY21580) are listed below:

Anti-CTLA4 heavy chain variable region (SEQ ID NO: 87):EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDWDDDKYYSTSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSY VYFDYWGQGTLVTVSSAnti-CTLA4 light chain variable region (SEQ ID NO: 100):DIQLTQSPSSLSASVGDRVTITCRASQSVRGRFLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSSWPPTFG QGTKVEIKR.

Pairs of plasmids were transiently transfected into HEK293F cells. Aftersix days, the supernatants were harvested, cleared by centrifugation andfiltration, and IgGs were purified with standard protein A affinitychromatography (MabSelect SuRe, GE Healthcare). The IgGs were eluted andneutralized, and buffer exchanged into PB buffer (20 mM sodiumphosphate, 150 mM NaCl, pH 7.0). Protein concentrations were determinedby UV-spectrophotometry, and IgG purity was analyzed under denaturing,reducing and non-reducing conditions by SDS-PAGE or SEC-HPLC.Importantly, the expression levels of the activatable antibodies inHEK293 cells were similar to their parental antibody, and theirpurification yields after protein A resin were also similar, suggestingthat the presence of the masking and cleavage peptides do not have anegative impact on antibody expression in mammalian cells.

Measurement of Masking Efficiency

The ForteBio Octet RED96 system (Pall, USA) was used to quickly assessthe efficiency of the masking peptides. Briefly, activatable antibodies(and their parent antibody, TY21580) were diluted to 30 μg/mL in KBbuffer (PBS buffer supplemented with 0.02% Tween 20 and 0.1% BSA), andcaptured by anti-Human IgG Capture (AHC) Biosensors (Pall, USA) inparallel. The sensors were then allowed to associate with His-taggedCTLA4 protein (25 nM) for 300 seconds, and then dissociate in KB bufferfor another 300 seconds. The association and dissociation curves werefitted to a 1:1 Langmuir binding model using ForteBio Data Analysis 7.1(Pall, USA) according to the manufacturer's guidelines. As shown inFIGS. 34A-B, the responses achieved with the activatable antibodies weresignificantly lower than that for the parent antibody, suggesting thatmasking peptides effectively blocked the binding of the antibody to itsantigen. Among the four activatable antibodies, however, TY22401 wasless effective, consistent with the results from the ELISA assaydiscussed below.

Recombinant human CTLA4-Fc was diluted to 1 μg/mL in PBS and coated on aMaxisorp plate at 4° C. overnight. Plates were blocked with PBSsupplemented with 3% non-fat milk at 37° C. for 1 hour. After washing,100 μL of 3-fold serial dilutions of antibodies were added to each well.After incubation at 37° C. for 1 hour, plates were washed four times,and 100 μL HRP conjugated anti-human IgG (Fab specific) (1:6000dilution) was added to each well. Plates were incubated at 37° C. for 1hour, washed four times, and then 50 μL TMB substrate solution was addedto each well, and the plate was incubated at room temperature.Absorbance at 450 nm was measured after the reactions were stopped with50 μL H₂SO₄ per well. The EC₅₀ was evaluated by fitting the ELISA datausing the asymmetrical sigmoidal (five-parameter logistic equation)model of GraphPad Prism 6 software. Experiments for activatableantibodies TY22401, TY22402, and TY22404 were performed twice, leadingto two calculated masking efficiencies being obtained for each of theseactivatable antibodies. Masking efficiencies for each activatableantibody were calculated by dividing the EC₅₀ for binding of theactivatable antibody by the EC₅₀ of the parental antibody (TY21580). Asshown in FIGS. 35A-B and Table 15, compared with the parental antibody,all of the activatable antibodies showed dramatically reduced binding toits antigen, and the calculated masking efficiency ranged from 48 to2213. Differences in masking efficiency likely resulted from variationin measurement and data fitting for the EC₅₀ values, and the maskingefficiency for each activatable antibody likely falls within thecalculated ranges (e.g., the masking efficiency for activatable antibodyTY22402 is between 377 and 2213). These results indicated that multiplemasking peptides identified from the CPLs maintained their maskingefficiency when expressed in mammalian cells, and as part of a full IgGmolecule.

TABLE 15 Activatable antibody ELISAs prior to protease cleavage MaskingSample EC₅₀ effi- ID: LogEC₅₀: M: nM: R2: ciency: Data Batch 1 TY21580−9.665 2.161E−10 0.216 0.999 1.0 TY22401 −7.623 2.382E−08 23.82 0.997110 TY22402 −6.321 4.779E−07 477.9 0.997 2213 TY22404 −6.749 178.4E−07178.4 0.998 826 Data Batch 2 TY21580 −9.478 3.324E−10 0.3324 0.998 1.0TY22401 −7.800 1.586E−08 15.86 0.994 48 TY22402 −6.902 1.254E−07 125.40.998 377 TY22404 −6.892 1.281E−07 128.1 0.998 385 TY21580 −9.48 3.3E−100.33 1.0 TY22563 −7.32 4.771E−08 47.71 143.5 TY22564 −7.41 3.898E−0838.98 117.3 TY22565 −6.68 2.099E−07 209.9 631.5 TY22566 −6.79 1.6264E−07162.6 489.2Removal of the Masking Peptide Restores Antibody Activity

The purified activatable antibodies were treated with the proteaseswhich recognize the cleavage sequences, and were then tested todetermine whether removal of the masking peptide restored theiractivity. As an example, 20 μg of TY22404 (0.5 mg/mL) was treated with 1μg of recombinant human uPA (Acrobiosystems, #PLU-H5229) in reactionbuffer (50 mM Tris-HCl, 0.01% Tween 20, pH 8.5); or TY22404 was treatedwith 5 or 10 units of recombinant human MMP-9 (BioVision, #7867-500) inreaction buffer (50 mM Tris, 150 mM NaCl, 5 mM CaCl₂, 20 μM ZnCl₂, pH7.5). The reactions were carried out at 37° C. for 21 hours. The maskingpeptides were confirmed to be removed from the light chain by SDS-PAGEanalysis FIG. 36A. The masking efficiency was then measured by ELISA asdescribed above. As shown in FIG. 36B and Table 16, after removal ofmasking peptide, the activatable antibody became indistinguishable fromthe parent antibody in its binding to the antigen.

TABLE 16 Activatable antibody ELISAs after protease cleavage EC₅₀ SampleID: LogEC₅₀: nM: Masking efficiency: TY21580 −9.35 0.447 1.0 TY22404−7.01 96.8 216 TY22404-uPA −9.40 0.402 0.9 TY22404-MMP-9 −9.39 0.412 0.9Activatable Antibody Developability Profiles

For manufacturing purpose, it is critical that the discoveredactivatable antibodies have a good developability profile. Severaldifferent tests were performed with purified activatable antibodies thatwere expressed in mammalian cells. The activatable antibodies wereadjusted to 1 mg/mL in 20 mM Histidine, pH 5.5, and antibody qualityanalysis was performed using analytical size-exclusion chromatographyusing a Waters 2695 with a Waters 2996 UV detector and aTSKgel g3000SWXL column (300 mm×7.8 mm) (Tosoh Bioscience). For each assay, 10 μg ofantibody was injected, and fractionation was performed at a flow rate of0.5 mL/min in buffer (200 mM sodium phosphate at pH 7.0).

Three accelerated stress tests were conducted: incubation of theactivatable antibodies at 50° C. for 7 days, incubation of theactivatable antibodies at 40° C. for 28 days, and six cycles offreeze-thaw. The freeze-thaw tests were conducted by freezing 100 μLsample (1 mg/mL in 20 mM histidine, pH 5.5) at −80° C. for 30 minutes,followed by thawing at room temperature for 60 min. As shown in FIGS.37A-C, all activatable antibodies remained stable, and exhibited littleaggregation after storage at 50° C. for 7 days or 40° C. for 28 days.After six cycles of freeze-thaw, they showed slight deterioration;however, the main monomer peak remained around 95%, indicating thatthese activatable antibodies were very stable under these acceleratedstress tests. Without wishing to be bound by theory, it is worth notingthat the activatable antibodies had not yet gone through an extensivebuffer optimization process, and therefore, the stability of theactivatable antibodies may be further improved with optimized buffer andexcipient.

Next, activatable antibodies were concentrated to more than 150 mg/mL in20 mM histidine, pH 5.5 (Table 17). No activatable antibodyprecipitation was observed, and viscosity of the samples was quitemanageable. The concentrated activatable antibodies were then diluted toeither 20 mg/mL or 1 mg/mL for analysis of high molecular weight (HMW)species. As shown in FIG. 38 and Table 17, no apparent increase of theHMW species was observed, suggesting that these activatable antibodieswere very soluble and stable in the buffer tested, up to highconcentrations.

TABLE 17 Concentration of activatable antibodies >150 mg/mL Sample ID:Starting conc. (mg/mL): High conc. (mg/mL): TY22401 10.9 187.2 TY224028.4 160.0

To study the stability of the activatable antibodies at low pH, thepurified activatable antibodies (at 10 mg/mL in 20 mM histidine, pH 5.5)were titrated to 1 mg/mL with citric acid, and the pH was adjusted to3.7 and held at room temperature for 30 and 60 minutes. Afterwards, thesamples were neutralized to pH 7.0 with 1 M Tris-base. The maskingefficiency of the activatable antibodies was measured with ForteBio, asdescribed above. As shown in FIG. 39, masking efficiency remainedunchanged after low pH incubation for 30 or 60 minutes, suggesting thatthe masking peptides retained their blocking efficacy after low pHincubation.

Taken together, the data indicates that the discovered activatableantibodies remained stable under various stress conditions, andtherefore, they have good developability profile.

Example 9: In Vitro and In Vivo Characterization of ActivatableAntibodies Targeting CTLA4

It is known that CTLA-4 activity on T cells is related to the first(TCR/CD3) and second signals involving B7-CD28/CTLA-4.

In Vitro Functional Characterization

Here the activities of the activatable antibodies targeting CTLA4 wereevaluated in the presence of a low concentration of anti-CD3 antibody onhuman PMBC activation. Human PBMCs were freshly isolated from the bloodof a healthy donor (#44) by density gradient centrifugation usingHistopaque-1077 (Sigma). Anti-CD3 (OKT-3) antibody was coated on a 96well plate overnight at 4° C. After washing, 1×10{circumflex over ( )}5freshly isolated human PBMCs were added to each well, followed by theaddition of the test articles at different concentrations. Induction ofIL-2 was measured 48 hours after stimulation using a Human IL-2 ELISAReady-SET-Go (Invitrogen) kit. IFN-γ in the supernatant was measuredusing a Human IFN-γ ELISA Ready-SET-Go (Invitrogen) kit. As demonstratedin FIGS. 40A-B, at high concentrations, TY22404 induced IL-2 production,and TY22401 induced IFN-γ production. Nevertheless, the activities ofthe activatable antibodies were significantly lower than that of theparental TY21580 antibody.

Next, the antibody-dependent cell cytotoxicity activities of theactivatable antibodies were tested and compared with that of theparental antibody TY21580. An ADCC reporter gene assay was used toevaluate the ADCC activities of the activatable antibodies. HEK293Fcells overexpressing human CTLA4 (HEK293F/hCTLA-4 cells) were used astarget cells; a Jurkat cell line overexpressing CD16a and NFAT-Luc(Jurkat/CD16a cells) was used as effector cells. 1×10{circumflex over( )}5 Jurkat/CD16a cells and 1×10{circumflex over ( )}4 HEK293F/hCTLA-4cells (E:T ratio 10:1) were mixed with different concentrations ofantibody. After incubation for 6 hours, 100 μL of One-Glo reagent wasadded to the cells, and the cells were lysed for 10 min. Supernatantswere removed for luminescence measurements using a SpectraMax i3x platereader. As shown in FIG. 41, the activatable antibodies showed severallog lower ADCC activities than the parental antibody TY21580. The ADCCactivity of TY22401 was higher than that of TY22402 and TY22404. Takentogether, the in vitro data indicates that the better masked activatableantibodies had less ADCC activity.

The anti-tumor activities of the activatable antibodies were nextevaluated and compared with the anti-tumor activity of the parentalantibody TY21580 in multiple syngeneic mouse tumor models, including anMC38 colorectal tumor model, a CT26 colorectal tumor models, an H22liver tumor model, and a 3LL lung tumor model.

Anti-Tumor Efficacy in an MC38 Colorectal Tumor Model

C57BL/6 mice (n=8 per group, female, 6-8 weeks old) were inoculatedsubcutaneously with MC38 (NTCC-MC38) murine colon cancer cells. Whentumors were established (70 mm3), treatment began with isotype controlantibody, parental antibody TY21580, or one of three activatableantibodies by intraperitoneal injection, twice a week. Tumor growth wasmonitored twice a week, the mean tumor volume±s.e.m. over time (FIG.42A) and individual tumor growth curves (FIG. 42B) were assessed. Asshown in FIGS. 42A-B, all three activatable antibodies showed potentanti-tumor activities, comparable to the parental antibody TY21580 inthe MC38 syngeneic mouse tumor model.

Anti-Tumor Efficacy in a CT26 Colorectal Tumor Model

BALB/c mice (n=8 per group, female, 7-8 weeks old) were inoculatedsubcutaneously with CT26 (Shanghai Institutes for Biological Sciences)murine colon cancer cells. When tumors were established (100 mm3),treatment began with isotype control antibody, parental antibodyTY21580, or one of three activatable antibodies at 5 mg/kg byintraperitoneal injection, twice a week. Tumor growth was monitoredtwice a week and reported as the mean tumor volume±s.e.m. over time. Asshown in FIG. 43, all three activatable antibodies showed potentanti-tumor activities, comparable to the parental antibody TY21580 inCT26 syngeneic mouse tumor model.

Anti-Tumor Efficacy in an H22 Liver Tumor Model

BALB/c mice (n=8 per group, female, 7-8 weeks old) were inoculatedsubcutaneously with H22 (China Center for Type Culture Collection)murine liver cancer cells. When tumors were established (100 mm3),treatment began with isotype control antibody, parental antibodyTY21580, or one of three activatable antibodies at 5 mg/kg byintraperitoneal injection, twice a week. Tumor growth was monitoredtwice a week and reported as the mean tumor volume±s.e.m. over time. Asshown in FIG. 44, all three Activatable antibodies showed potentanti-tumor activities, comparable to the parental antibody TY21580 inH22 syngeneic mouse tumor model.

Anti-Tumor Efficacy in a 3LL Lung Cancer Model

C57BL/6 mice (n=10 per group, female, 6-8 weeks old) were inoculatedsubcutaneously with 3LL (JCRB) murine lung cancer cells. When tumorswere established (75 mm3), treatment began with isotype controlantibody, parental antibody TY21580, or one of three activatableantibodies by intraperitoneal injection, twice a week. Tumor growth wasmonitored twice a week, the mean tumor volume±s.e.m. over time (FIG.45A) and individual tumor growth curves (FIG. 45B) were assessed. Asshown in FIGS. 45A-B, all three activatable antibodies showed potentanti-tumor activities, comparable to the parental antibody TY21580 in3LL syngeneic mouse tumor model.

Pharmacokinetic Analysis

A pharmacokinetics study was conducted in BALB/c female mice at abouteight weeks of age. Three mice per group were intraperitoneally injectedwith the test article at 10 mg/kg. Blood samples (˜50 ul per sample)were collected at 3, 6, 24, 48, 96, and 168 hours post-dosing. Blankcontrol blood was collected from three naïve female mice withoutantibody administration. Serum concentrations of each test antibody weredetermined by ELISA, in which anti-human IgG Fc was used for capture,and HRP-labeled anti-human IgG (Fab specific) antibody (Sigma) was usedfor detection (FIGS. 46A-C). As compared to the previous data collectedfor parental antibody TY21580, activatable antibodies TY22401 (FIG.46A), TY22402 (FIG. 46B), and TY22404 (FIG. 46C) had a much slowerclearance time and longer half-life. TY22401 has a half-life of 196hours, and the drug concentration at 168 hours was about 55 μg/mL.TY22402 had a half-life of 134 hours, and the drug concentration at 168hours was about 40 μg/mL. TY22404 had a half-life of 254 hours, and thedrug concentration at 168 hours was about 45 μg/mL. In comparison, theparental antibody TY21580 had a half-life of 107 hours, and the drugconcentration at 168 hours was about 17 μg/mL.

Repeated Dosing Toxicity Studies

While evaluating the effect of TY21580 on diabetes onset age in NODmice, it was found that high dosages of TY21580 could lead to animaldeath of NOD but not normal BALB/c mice. Here the NOD mouse model wasused to evaluate the safety of the activatable antibodies, as comparedto that of TY21580. NOD mice (n=5 per group, female, 6 weeks old) weretreated with isotype control antibody, parental antibody TY21580, or oneof three activatable antibodies by intraperitoneal injection at 50 mg/kgon days 0, 3, 7, and 12. In the TY21580 treatment group, 1 animal diedafter the third dosing, and 3 animals died after the fourth dosing. Asshown in FIG. 47, all animals treated with the isotype control or any ofthe three activatable antibodies were alive and in good health at thetermination of the study. These data indicated that the activatableantibodies have acceptable safety/toxicity profiles in mice, and, in NODmice, the activatable antibodies are much safer than the parentalantibody TY21580.

Example 10: Additional In Vivo Characterizations of ActivatableAntibodies Targeting CTLA4

In the previous studies of the parental antibody TY21580, it was foundthat repeated dosing of TY21580 lead to increased spleen size in bothfemale and male normal BALB/c mice. Other than that, TY21580 did notshow any significant side effects on other evaluated parameters,including the weights of many organs, liver histopathology, hematology,and blood biochemistry. Therefore, the effect of several activatableantibodies on spleen size was evaluated and compared with that of theparental antibody TY21580.

Repeated dosing toxicity of the activatable antibodies was conducted innormal BALB/c mice as follows: 50 mg/kg of isotype control antibody,TY21580 parental antibody, or activatable antibody TY22402, TY22566, orTY22401 was administered intraperitoneally (10 mL/kg) on days 1, 4, 7,and 11. Five female mice (five weeks old) were included in each group.Mice were monitored daily for abnormal behaviors and symptoms, andmeasured daily for food intake and body weight. On day 14, animals wereeuthanized for post-mortem examination and other analyses.Interestingly, while administration of activatable antibodies TY22402(FIG. 48A) or TY22566 (FIG. 48B) slightly increased the spleen size inmice as compared to the isotype control, these activatable antibodiesshowed significantly less effect on spleen size as compared toadministration of the parental antibody TY21580. Administration ofactivatable antibody TY22401 significantly increased the spleen size ascompared to isotype control, but still to a lesser extent than wasobserved using the parental antibody TY21580 (FIG. 48C).

As CTLA4 is constitutively expressed on Treg cells, the effects ofactivatable antibodies TY22402, TY22566, or TY22401 on Treg cells, CD4⁺T cells, and CD8⁺ T cells in both whole blood and the spleen wereevaluated and compared to parental antibody TY21580. Monocytes fromwhole blood or splenocytes were stained and gated using the followingantibodies: anti-CD45-BV421, anti-CD3-AF488, anti-CD4-BV510,anti-CD8a-PerCP-cy5.5, anti-CD25-APC, and antiFoxP3-PE. Treg cells weredefined as CD45⁺CD3⁺CD4⁺CD25⁺Foxp3⁺. As shown in Table 18, compared toisotype control, parental antibody TY21580 increased the percentage ofTreg cells in the spleen; however, activatable antibodies TY22402 andTY22566 did not affect the percentage of splenic Tregs. In whole blood,TY21580, TY22402, and TY22566 slightly increased the percentage of Tregcells when compared to isotype control. Activatable antibody TY22401increased the percentage of Treg cells in the spleen and whole blood.The percentages of CD4⁺ and CD8⁺ T cells were not significantly alteredby TY21580, TY22402, TY22566, or TY22401 (data not shown).

TABLE 18 FACS analysis showing effect of activatable antibodies onspleen Treg and blood Treg cells Spleen: Treg % in Blood: Treg % inGroup: Sample: CD4⁺ T cells CD4⁺ T cells Isotype control 6-1 10.50 1.71(50 mg/kg, BIW) 6-2 8.25 0.70 6-3 8.04 0.67 6-4 6.81 0.90 Mean 8.40 1.00SD 1.54 0.49 TY21580 5-1 11.55 2.14 (50 mg/kg, BIW) 5-2 8.52 1.56 5-310.64 1.84 5-4 11.69 1.40 Mean 10.60 1.74 SD 1.47 0.32 TY22402 3-1 7.101.42 (50 mg/kg, BIW) 3-2 7.08 1.00 3-3 10.77 1.70 3-4 10.25 1.98 Mean8.80 1.53 SD 1.99 0.42 TY22566 4-1 11.31 3.07 (50 mg/kg, BIW) 4-2 6.421.04 4-3 11.31 2.70 4-4 5.26 1.48 Mean 8.58 2.07 SD 3.19 0.97 TY224012-1 10.39 2.23 (50 mg/kg, BIW) 2-2 11.12 4.09 2-3 10.63 1.71 2-4 11.962.76 Mean 11.03 2.70 SD 0.69 1.02

Example 11: Additional Activatable Antibody Developability Assays

Several different tests were performed with purified activatableantibodies that were expressed in mammalian cells to determine theirdevelopability profiles. Three accelerated stress tests were conducted.First, activatable antibodies TY22401, TY22402, or TY22566 wereincubated at 50° C. for 7 days, and their stabilites were determined bySEC and compared to isotype control (FIG. 49). Only slight increases, ifany, in high molecular weight (HMW) aggregates or low molecular weight(LMW) fragments were observed for the activatable antibodies afterincubation for 7 days at 50° C. (Table 19).

TABLE 19 Activatable antibody stability at 50° C. for 7 days HMW % LMW %HMW % LMW % Sample: (control) (control) (50° C., 7 days) (50° C., 7days) TY22566 1.35 0.33 1.24 2.78 TY22401 2.15 0.20 2.13 1.97 TY224022.46 0 0.45 2.57

Nest, activatable antibodies TY22401, TY22402, or TY22566 were incubatedat 40° C. for 7, 14, 21, or 28 days, and their stabilites weredetermined by SEC and compared to isotype control (FIG. 50). Only slightincreases, if any, in high molecular weight (HMW) aggregates or lowmolecular weight (LMW) fragments were observed for the activatableantibodies after incubation for at 40° C. at the various time points(Table 20).

TABLE 20 Activatable antibody stability at 40° C. for 28 days HMW % LMW% HMW % LMW % Sample: (control) (control) (40° C., 28 days) (40° C., 28days) TY22566 1.02 0.00 1.86 1.64 TY22401 1.57 0.00 2.03 1.19 TY224021.02 0.00 1.86 1.64

In addition, activatable antibodies TY22401, TY22402, or TY22566 weresubjected to six cycles of freeze-thaw. The freeze-thaw tests wereconducted by freezing 100 μL sample (1 mg/mL in 20 mM histidine, pH 5.5)at −80° C. for 30 minutes, followed by thawing at room temperature for60 min, and stability was measured by SEC and compared to isotypecontrol (FIG. 51). Only slight increases, if any, in high molecularweight (HMW) aggregates were observed for the activatable antibodiesafter these freeze-thaw cycles (Table 21).

TABLE 21 Activatable antibody stability after 6 freeze-thaw cycles HMW %HMW % Sample: (control) (6 cycles) TY22566 1.35 4.36 TY22401 2.15 4.96TY22402 2.46 3.48

Next, activatable antibodies were concentrated to more than 115 mg/mL in20 mM histidine, pH 5.5. The concentrated activatable antibodies werethen diluted to 20 mg/mL for analysis of high molecular weight (HMW)species. As shown in FIG. 52 and Table 22, no apparent increase of theHMW species was observed, suggesting that these activatable antibodieswere very soluble and stable in the buffer tested, up to highconcentrations.

TABLE 22 Concentration of activatable antibodies >150 mg/mL ConcentratedHMW % HMW % Sample: (mg/mL) (control) (concentrated) TY22566 125.73 2.353.69 TY22401 115.98 1.47 1.80 TY22402 128.87 1.82 4.04

Taken together, the data indicates that the discovered activatableantibodies remained stable under various stress conditions, andtherefore, even without formulation optimization, they have gooddevelopability profiles (FIG. 53).

Example 12: Epitope Binding and Cross-Reactivity of TY21580 andIpilimumab to CTLA4

The F sheet, FG loop, and G sheet of human CTLA4 contain most of thecontact residues important for CTLA4 interaction with its ligands CD80and CD86 (FIG. 54; FIGS. 55A and 55B). For example, the humanCTLA-4/CD86 interface is formed by residues E33, R35, T53, E97, M99,Y100, P101, P102, P103, Y104, Y105, L₁₀₆ on the front β-sheet of CTLA4(Schwartz et al. (2001) Nature 410(6828): 604-608) and alaninesubstitutions of residues in the FG loop of CTLA4 (99MYPPPYY105) reduceor abolish binding to CD80 (Stamper et al. (2001) Nature 410(6828):608-611).

Ipilimumab, an anti-CTLA4 antibody, also interfaces with CTLA4 on thefront β-sheet of CTLA4 at the F and G strands (residues I93, K95, E97,L106, and I108), the FG loop (residues 99MYPPPY104) and the CC′ loop(residues L39, V46, and I93; located opposite the FG loop). A perviousstudy showed that mutation of residues S20, R35, R40, Q76, D88, K95,E97, Y104, L106, and I108 of human CTLA4 resulted in significant loss ofIpilimumab binding, with mutations in residues R35, K95, E97, Y104, andI108 showing a severe loss in CTLA4 binding. In addition, Ipilimumab wasshown to directly contact residues R35, K95, E97, Y104, L106, and I108of CTLA4 in a Ipilimumab-CTLA4 crystal structure (Ramagopal et al.(2017) Proc Natl Acad Sci USA 114(21): 4223-4232). In addition, crystalstructures have revealed that the Ipilimumab epitope partially occupiesthe CD80/CD86 binding site of CTLA4, with a larger interface area (1,880Å2 for CTLA4/Ipilimumab) than the receptor-ligand interfaces (1,255 Å2for CTLA-4/CD80 and 1,212 A2 for CTLA-4/CD86) (Ramagopal et al. (2017)Proc Natl Acad Sci USA 114(21): 4223-4232; Lee et al. (2016) Nat Commun7(13354); He et al. (2017) Oncotarget 8:67129-67139).

The epitope binding characteristics of the anti-CTLA4 antibodies TY21580and Ipilimumab were investigated. A systematic approach was taken tonarrow in on the important CTLA4 epitope motifs and residues for TY21580and Ipilimumab binding to CTLA4. First, a low copy number, CEN/ARS-basedvector was used to express mouse CTLA4, human CTLA4, or various humanCTLA4 mutant proteins under the control of the inducible GAL1-10promoter for cell surface display in the yeast S. cerevisiae (Boder andWittrup (1997) Nat Biotechnol 15(6):553-7). In mutant CTLA4 vectors,particular motifs or residues of human CTLA4 were mutated to alanine orto the corresponding residues of the mouse CTLA4. TY21580 or Ipilimumabwas added to the cells displaying CTLA4 and antibody binding wasassessed through flow cytometry.

For comparison, the contact residues on CTLA4 from CTLA4-CD80(PDBID:1I8L), CTLA4-CD86 (PDBID:1I85), and CTLA4-Ipilimumab (PDBID:5XJ3, 5TRU) crystal structures (within 4 Å of C-alpha atoms from theinter-molecular interface) are shown in FIG. 54. The contact residuesdefined by their interacting C-alpha atoms are marked as thegray-colored residues in their human sequences derived from theircorresponding X-ray structures.

As shown in FIGS. 56A and 56B, the difference in the cross-reactivity ofTY21580 and Ipilimumab are striking, with TY21580 binding to both humanand mouse CTLA4 and Ipilimumab binding to human CTLA4 but not mouseCTLA4. For comparison, human CD80 (FIG. 56C), human CD86 (FIG. 56D), andmouse CD86 (FIG. 56E) interact with both human and mouse CTLA4.

To further investigate the epitope binding profiles of Ipilimumab andTY21580 to CTLA4, various motifs of human CTLA4 were mutated andassessed for binding. TY21580, Ipilimumab, human CD80, human CD86, andmouse CD86 maintained a similar binding ability to the ADS42AAA CTLA4mutant as compared to wild-type CTLA4 (FIG. 56A-56E). However, when thehuman CTLA4 CC′ loop motif (42ADSQVT47) was mutated to the mouse CTLA4CC′ loop motif (42TNDQMT47), TY21580 bound two times stronger to themutant compared to Ipilimumab, human CD80, human CD86, and mouse CD86binding to the mutant (FIG. 56A-56E). It is consistently observed thatCD80 and CD86 do not directly bind to this CC′ loop motif (see e.g.,FIG. 54). Although Ipilimumab is in direct contact with this motif inhuman CTLA4 (FIG. 54; Ramagopal et al. (2017) Proc Natl Acad Sci USA114(21): 4223-4232), the impact of human CTLA4 binding upon mutationalchange of the CC′ loop is minimal (see human WT vs. ADSQVT42TNDQMT inFIG. 56B). Taken together, these results suggest that the CC′ loop motifdoes not contribute significantly to the binding affinity of Ipilimumabor TY21580 to human CTLA4.

One of the most interesting observations from the epitope mapping datais the striking difference between Ipilimumab and TY21580 binding toCTLA4 when mutations are introduced from residues 105 to 108 in humanCTLA4. For example, when YL105AA and LGI106AAA were introduced intohuman CTLA4, Ipilimumab lost its ability to bind CTLA4 (FIG. 56B),consistent with the known contact amino acids important for Ipilimumabbinding to human CTLA4, shown in FIG. 54. In addition, as shown in FIGS.56A and 56C-56E, the binding affinity of TY21580, human CD80, humanCD86, and mouse CD86 to the CTLA4 YL105AA mutant was weakened. While thebinding affinity of human CD80, human CD86, and mouse CD86 withLGI106AAA was also weakened, the binding affinity of TY21580 withLGI106AAA was significantly enhanced. This is in contrast to the totalloss of binding affinity of Ipilimumab with LGI106AAA.

For epitope mapping of the binding sites of CTLA4 by TY21580 andIpilimumab, mutations were introduced into specific sites of humanCTLA4. Sites that were selected for mutagenesis were known contact sitesof CTLA4 by Ipilimumab, CD80, and CD86 (based on their crystalstructures, see FIG. 54) which had large sequence variations betweenhuman and mouse CTLA4 sequences. For example, FIGS. 55A and 55B show thefront view of the complex structures of CD80 and CD86 with human CTLA4,with I108 highlighted and shown as a ball. I108 is one of the mostcritical sites in contact with Ipilimumab, however it is far away fromthe CD80 and CD86 contact regions. The G strand contains the majority ofthe residues contacting Ipilimumab, CD80, and CD86 that differsignificantly between human and mouse CTLA4, as shown in FIG. 54. TheCC′ loop motif also contains contact residues with Ipilimumab that aredivergent between human and mouse CTLA4, however, the results in FIGS.56A-56E indicated that the mutants introduced into CC′ loop motif do notsignificantly contribute to overall binding of Ipilimumab, TY21580,CD80, or CD86 to CTLA4. Thus, to further assess the binding contributionof the G strand motif to Ipilimumab, TY21580, CD80, and CD86, the Gstrand of human CTLA4 (105YLGIG109) was mutated to mouse CTLA4(105FVGMG109).

As shown in FIG. 56B, Ipilimumab suffered a severe loss of bindingaffinity to the YLGIG105FVGMG mutant. In contrast, TY21580, human CD80,human CD86, and mouse CD86 retained binding ability when this mutationwas introduced (FIGS. 56A, 56C-56E). This indicates that TY21580 has adifferent binding epitope from Ipilimumab due to this difference in Gstrand binding between human and mouse CTLA4.

In addition, other amino acids in the G strand of human CTLA4 werereplaced by the corresponding mouse sequences or other amino acids toprobe their interactions with Ipilimumab, TY21580, CD80 and CD86. I108is known to an extremely important amino acid for Ipilimumab binding.Consistent with this expectation, Ipilimumab lost its binding affinityto I108A, I108S, I108E, I108K and I108R CTLA4 mutants (FIG. 56B). Incontrast, I108 mutants (I108A, I108S, I108E, I108K and I108R) had noimpact on the binding affinity of TY21580, CD80 or CD86, highlightingthe important differences in the binding epitope of TY21580, CD80, andCD86 compared to Ipilimumab, as shown by I108, one of the most criticalbinding sites of Ipilimumab (Ramagopal et al. (2017) Proc Natl Acad SciUSA 114(21): 4223-4232). The complex structure in FIGS. 55A and 55B showthat I108 is indeed physically distant from the binding sites betweenCTLA4 and its ligands CD80 and CD86, and thus may not have a significantimpact on binding between CD80/CD86 and CTLA4. Our observation confirmsthat TY21580 is more similar in its binding epitope with CD80/CD86,differentiated from the epitope by Ipilimumab. Therefore, TY21580 isthought to specifically bind to an epitope comprising amino acidresidues Y105 and L106, but not I108 of human CTLA4.

Example 13: Effect of TY21580 and Ipilimumab on CTLA4 Ligand BindingBlockade

TY21580 and Ipilimumab were tested for their ability to block binding ofCTLA4 to its cognate ligands CD80 and CD86 through ELISA-basedexperiments, as described in Example 3 (Ligand competition binding byELISA). Briefly, in one experiment, ELISA microplates were coated withrecombinant human CTLA4 proteins (1 μg/mL) and biotinylated CTLA4ligands (CD80 at 1 μg/mL or CD86 at 2 μg/mL) were added to each wellalong with serial dilutions of either TY21580, Ipilimumab, or an isotypecontrol antibody (FIGS. 57A and 57B). In another experiment, ELISAmicroplates were coated with recombinant CD80 or CD86 protein (1 μg/mL)and biotinylated human CTLA4 (0.2 μg/mL or 1 μg/mL) was added to eachwell along with serial dilutions of either TY21580, Ipilimumab, or anisotype control antibody (FIGS. 57C and 57D). In both experiments, thelevel of binding between human CTLA4 and either CD80 or CD86 wasdetected using HRP-labeled Neutravidin as described in Example 3 (Ligandcompetition binding by ELISA).

As shown in FIGS. 57A and 57B, when CTLA4 was immobilized onto themicroplate, TY21580 and Ipilimumab blocked CD80 and CD86 binding toCTLA4 in a dose-dependent manner, whereas the isotype control antibodyshowed no blocking activity, indicative of assay specificity. As shownin FIGS. 57C and 57D, when CD80 or CD86 was immobilized onto themicroplate, TY21580 and Ipilimumab again blocked CD80 and CD86 bindingto CTLA4 in a dose-dependent manner, while the isotype control antibodydisplayed no blocking activity. As shown in Table 23, although TY21580and Ipilimumab both block ligand binding to CTLA4, the dose-dependentblocking activities of TY21580 and Ipilimumab were strikingly differentwhen human CTLA4 was immobilized onto the microplate compared to whenCD80 or CD86 was immobilized onto the microplate. These results indicatethat TY21580 and Ipilimumab exhibit comparable ligand blockingactivities with similar IC50s under CTLA4 immobilization conditions.However, under CD80 or CD86 ligand immobilization conditions, Ipilimumabexhibited a much stronger ligand blocking activity than TY21580 for bothCD80 and CD86, as shown in FIGS. 57C and 57D and Table 23. However,additional experiments suggested that a complete blockade of CTLA4'sinteraction with its ligands upon adding an anti-CTLA4 antibody at asaturating concentration is not necessary for tumor rejection, meaningthat TY21580 could still activate T cells and have potent anti-tumorefficacy without completely blocking CD80 or CD86. In fact, TY21586showed complete blockage of CD80 and CD86, similar to Ipilimumab, asshown in FIG. 58. However, TY21586 and Ipilimumab have much lower ADCCreporter activity than other antibodies tested (FIG. 59), and aretherefore less effective in depleting Treg cells via the ADCC effect(see also Tang et al. (2018) Cell Biosci. 8(30):1-3). Without wishing tobe bound by theory, it is believed that CD28-dependent T cell activation(e.g., by blocking CTLA4 activity) is important for effective tumorrejection, however overstimulation of T cells can be detrimental. Thus,the weaker ligand blocking observed for TY21580 may be an advantage overthe stronger ligand blocking activity of Ipilimumab.

TABLE 23 IC50s of TY21580 and Ipilimumab for CTLA4 ligand bindingblockade Test Plate-bound CTLA4 CTLA4 in solution antibody CD80 in CD86in Plate-bound Plate-bound IC₅₀ (nM) solution solution CD80 CD86 TY215803.809 0.4806 59.08 5.583 Ipilimumab 4.116 0.5873 1.789 1.429

Although the underlying mechanisms for these differences are currentlyunclear, the results indicate that there are inherent differencesbetween the properties of TY21580 and Ipilimumab. Taken together, theseresults suggest that TY21580 can act as a ligand blocker by disruptingCD80 and CD86 binding to its inhibitory receptor CTLA4. Without wishingto be bound by theory, it is thought that relieving CD80 and CD86 fromCTLA4 sequestration through weak ligand blocking allows these ligands tosignal through co-stimulatory receptor CD28 during T cell activation,which can lead to increased effectiveness and safety during T cellimmune activation.

Example 14: Effect of TY21580 and Ipilimumab on CTLA4 Blockade of CD28Pathway Activation

Using a cell-based CTLA4 blockade bioassay developed by Promega (CAT#JA3001 and JA3005), the CTLA4 blocking functions of TY21580 andIpilimumab were assessed. This assay involved co-culturing twogenetically engineered cell lines in the presence or absence of TY21580or Ipilimumab, then measuring the bioluminescent signal generated by aluciferase reporter gene using the Bio-Glo™ Luciferase Assay System. Thetwo cell lines that were co-cultured were (1) CTLA4 effectorcells—Jurkat T cells expressing human CTLA4 and a luciferase reporterdriven by a native promoter that responds to TCR/CD28 activation; and(2) aAPC/Raji cells—Raji cells endogenously expressing CTLA4 ligandsCD80 and CD86 and additionally expressing a cell surface proteindesigned to activate TCRs in an antigen-independent manner. When thesetwo cell types are co-cultured, CTLA4 competes with CD28 for CD80 andCD86 binding, which inhibits activation of the CD28 pathway and leads tolow promoter-mediated luminescence. The addition of anti-CTLA4antibodies to this system may block the interaction of CTLA4 with itsligands CD80 and CD86, resulting in higher TCR/CD28 activation andsubsequent promoter-mediated luminescence.

As shown in FIG. 58, all tested antibodies activated the TCR/CD28pathway in a dose-dependent manner, as evidenced by the increasingreporter gene signal (i.e., luminescence) as antibody concentrationincreased. In comparison, the isotype control antibody showed noactivity. These results demonstrate a range of CTLA4 functional blockingactivities by these different anti-CTLA4 antibodies. Although the EC50sof the antibodies were somewhat similar (Table 24), the magnitude offunctional signaling activation stimulated by these antibodies indicatedthat Ipilimumab and TY21586 were the most potent CTLA4 blockers tested,followed by TY21680, TY21580, and TY21687 (FIG. 58). TY21691 had theweakest activity as a CTLA4 blocker out of the antibodies tested. Theseresults demonstrate that all of the tested antibodies can block theinteraction of CTLA4 with CD80 and CD86, resulting in downstreamTCR/CD28 pathway activity in a dose-dependent manner. Without wishing tobe bound by theory, the results suggest that all of the testedantibodies may increase T cell activation, which could be advantageousfor the treatment of cancer. As discussed above, weaker ligand blockerssuch as TY21580 may be particularly effective in treating cancer becauseof their reduced risk of overstimulating T cells compared to strongbinders.

TABLE 24 EC50s of anti-CTLA4 antibodies for TCR/CD28 pathway activationIpilimumab TY21580 TY21586 TY21687 TY21680 TY21691 EC₅₀ (μg/mL) 1.625.06 4.66 5.01 8.21 2.64

Example 15: ADCC Activity of Anti-CTLA4 Antibodies

Antibody-dependent cell cytotoxicity (ADCC) activities of Ipilimumab,TY21580, TY21586, TY21687, TY21680, and TY21691 were tested andcompared. An ADCC reporter gene assay was used to evaluate the ADCCactivities of the activatable antibodies. HEK293F cells overexpressinghuman CTLA4 (HEK293F/hCTLA4 cells) were used as target cells; a Jurkatcell line overexpressing CD16 and NFAT-Luc (Jurkat/CD16a cells) was usedas effector cells. 1.2×10⁵ Jurkat/CD16a cells and 2×10⁴ HEK293F/hCTLA4cells (E:T ratio 6:1) were mixed in a 96-well tissue culture microplatein the presence or absence of serially diluted anti-CTLA4 antibodies.After incubation for 6 hours, One-Glo reagent was added to the cells,and the cells were lysed. To measure reporter gene activity,supernatants were removed for luminescence measurements using aSpectraMax i3x plate reader. A human IgG1 isotype control antibody wasused as a negative control.

As shown in FIG. 59, all tested antibodies showed various degrees ofADCC signaling activation in a dose-dependent manner, whereas theisotype control showed no ADCC activity whatsoever. These resultsdemonstrate a range of ADCC signaling/stimulatory activities by thesedifferent anti-CTLA4 antibodies. As demonstrated by both the magnitudeof signaling activation (FIG. 59) and the EC50s (Table 25), TY21580induced the most potent ADCC signaling activity of all of the antibodiestested. TY21691 was the least active antibody in inducing ADCCsignaling. While earlier data demonstrated that these antibodies havesimilar binding affinities to human CTLA4, with KDs in the single digitnMs, these ADCC reporter results suggest that the binding epitopes ofthe tested antibodies likely impact ADCC activity more significantlythan their binding affinities. It should be noted that although TY21586,TY21680, TY21580, TY21687, and TY21691 are cross-reactive, the aboveresults show that their effectiveness in ligand blocking and ADCCactivity is dramatically different, suggesting that subtle differencesin antibody epitope binding sites may result in significant differencesin anti-tumor activities.

TABLE 25 EC50s of anti-CTLA4 antibodies for ADCC reporter signalingIpilimumab TY21580 TY21586 TY21687 TY21680 TY21691 EC50 (μg/ml) 1.080.174 4.69 9.5 6.76 >10

Example 16: Anti-Tumor Efficacy of TY21580 and Ipilimumab in a MurineMC38 Colorectal Tumor Model

To determine the anti-tumor efficacy of TY21580 and Ipilimumab, humanCTLA4 knock-in C57BL/6 mice (n=6 per group, female, 5-9 weeks old) wereinoculated subcutaneously with MC38 murine colon cancer cells. Whentumors were established (99 mm³), mice were treated with an isotypecontrol antibody (1 mg/kg), TY21580 (1 mg/kg or 0.2 mg/kg), orIpilimumab (1 mg/kg or 0.2 mg/kg) by intraperitoneal injection twice aweek for two weeks. Group averaged tumor growth (FIG. 60A) andindividual tumor growth of each mouse in different groups (FIG. 60B) wasmonitored twice a week and reported as the mean tumor volume±SEM overtime. As shown in FIGS. 60A and 60B, TY21580 showed a completeanti-tumor effect at doses of 1 mg/kg (Group-2) and 0.2 mg/kg (Group-3),and all tumors in these mice were completely abolished except for a verysmall tumor left at Day 32 post first dosing in one mouse. Ipilimumabalso showed a complete anti-tumor effect at the dose of 1 mg/kg(Group-4); however, half ( 3/6) of the tumors treated with the lowerdose of Ipilimumab (0.2 mg/kg, Group-5) escaped from tumor suppressionat Day 32 post first dosing. This shows that TY21580 is more efficaciousthan Ipilimumab in terms of anti-tumor activity. Both TY21580 andIpilimumab were well tolerated, no animals died during the study, and nosignificant body weight loss was observed in mice at the dose levelstested. In summary, these results suggest that TY21580 is a safe andeffective anti-cancer agent with more potent anti-cancer activity thanIpilimumab.

Example 17: Effect of TY21580 and Ipilimumab on Intra-Tumoral RegulatoryT (Treg) Cell Levels in a Murine MC38 Colon Cancer Model

The percentages of T regulatory (Treg) cells (CD4⁺CD25⁺) in CD4⁺ T cellsubpopulations after treatment with TY21580 or Ipilimumab were evaluatedin a subcutaneous MC38 murine colon cancer syngeneic model (FIG. 61A).Tumor-bearing animals were treated with TY21580 or Ipilimumab at 1 mg/kg(Q3d×3 doses). CD4⁺ T cells were isolated from tumors, then tumorinfiltrating lymphocytes (TILs) and peripheral cells (i.e., PBMCs andspleen cells) were isolated as subpopulations from these CD4⁺ T cells.

In the TILs, the percentage of Treg cells was significantly reducedafter TY21580 treatment (15.2% for TY21580 treatment group vs. 41.6% forisotype control group, P<0.001). There was no significant reduction ofTreg cells after Ipilimumab treatment (28.4% for Ipilimumab vs. 41.6%for isotype control, P=ns). This was consistent with the observationthat Ipilimumab does not significantly change or deplete FOXP3+ Tregcells within the microtumor environment (Sharma et al. (2018) Clin.Cancer Res. online publication only, PMID 30054281; Ferrara et al.(2018) Clin. Cancer Res.). In PBMCs, the percentage of Treg cells wasslightly increased after TY21580 treatment (6.2% for TY21580 group vs.3.9% for isotype control group, P=0.01), but this effect was not seenafter Ipilimumab treatment (4.8% for Ipilimumab group vs. 3.9% forisotype control group). See also Ha et al. PNAS (2019) 116(2):609-618.In the spleen cells, both TY21580 and Ipilimumab treatment had no effecton the percentage of Treg cells (8.6% for isotype control group; 7.8%for TY21580 group; 9.4% for Ipilimumab group).

The ratio of cytotoxic T lymphocytes (CD8⁺ T cells) to Treg cells (i.e.,the CD8⁺/Treg ratio) was also evaluated (FIG. 61B). In the TILs, theCD8⁺/Treg ratio increased after treatment with TY21580 (18.7 for TY21580vs. 3.7 for isotype control; P=0.0517, close to 0.05). The effect ofIpilimumab treatment on the CD8⁺/Treg ratio was not significant comparedto the isotype control and, at best, was weaker than the effect ofTY21580. In PBMCs and spleen cells, the CD8⁺/Treg ratio was notsignificantly changed after TY21580 or Ipilimumab treatment. Theseresults demonstrate that TY21580 exhibits activities that induce Tregcell depletion and increase the CD8⁺/Treg ratio specifically in tumorinfiltrating cells (i.e., TILs), but not in peripheral cells (i.e.,PBMCs and spleen cells).

The regulatory activity of the TY21580 anti-CTLA4 antibody on T cellsprovides a mechanistic understanding for TY21580's in vivo anti-tumorefficacy. Without wishing to be bound by theory, the results suggestthat TY21580 reduces immunosuppressive Treg activity and enhancescytotoxic T lymphocyte (CD8⁺ T cell) activity in the tumormicroenvironment to mediate anti-tumor responses. The quantitativedifferences between TY21580 and Ipilimumab in tumoral Treg depletion andin the CD8⁺/Treg ratio also shows that TY21580 exhibits betteranti-tumor activity than Ipilimumab in vivo.

Example 18: Effect of TY21580 on Intra-Tumoral Regulatory T (Treg) CellLevels in a Murine CT26 Colon Cancer Model

The percentages of T regulatory (Treg) cells in CD4⁺ T cellsubpopulations after treatment with TY21580 was evaluated in asubcutaneous CT26 murine colon cancer syngeneic model (FIG. 62A).Tumor-bearing animals were treated with TY21580 (5 mg/kg on days 0 and3). CD4⁺ T cells were isolated from tumors, then tumor infiltratinglymphocytes (TILs) and peripheral cells (i.e., PBMCs and lymph node (LN)cells) were isolated as subpopulations from these CD4⁺ T cells.

As shown in FIG. 62A, TY21580 significantly reduced the percentage ofTreg cells in TILs compared to the isotype control (12.6% for TY21580vs. 38.5% for isotype control, P<0.001). However, TY21580 did notinfluence the percentage of Treg cells in peripheral lymphocytes (i.e.,PBMCs and lymph node (LN) cells) as compared to the isotype control. Thepercentage of Treg cells in PBMCs was 5.8% after TY21580 treatment vs.4.9% after isotype control treatment (P>0.05). The percentage of Tregcells in lymph node cells was 8.1% after TY21580 treatment vs. 7.7%after isotype control treatment (P>0.05).

The ratio of cytotoxic T lymphocytes (CD8⁺ T cells) to Treg cells (i.e.,the CD8⁺/Treg ratio) was also evaluated. As shown in FIG. 62B, TY21580also significantly increased the CD8⁺ T/Treg ratio in TILs compared tothe isotype control (8.0% for TY21580 vs. 0.6% for isotype control,P<0.01). However, TY21580 had no significant influence on the CD8⁺T/Treg ratio in peripheral lymphocytes. In PBMCs, the CD8⁺ T/Treg ratiowas 5.3 after TY21580 treatment vs. 6.3 after isotype control treatment(P>0.05). In lymph node cells, the CD8⁺ T/Treg ratio was 4.9 afterTY21580 treatment vs. 4.7 after isotype control treatment (P>0.05).

In addition, the CTLA4 expression levels in Foxp3⁺ CD4⁺ Treg cells fromthe TY21580 treatment group were significantly lower than those of theisotype control group in TILs (8985.2 MFI for TY21580 vs. 20948.0 MFIfor isotype control; FIG. 63). However, CTLA4 expression levels were notchanged in PBMCs (2046.7 MFI for TY21580 vs. 2740.9 MFI for isotypecontrol) or lymph nodes (3062.0 MFI for TY21580 vs. 3247.9 MFI forisotype control). Treg cells in the TILs also displayed much higherCTLA4 expression levels than Treg cells in the peripheral cells (i.e.,PBMCs and LNs), and CTLA4 expression was significantly lower in TIL Tregcells after TY21580 treatment.

Taken together, these results demonstrate that TY21580 exhibitsactivities that induce Treg depletion and increase the CD8⁺/Treg ratiospecifically in tumor cells (i.e., TILs), but not in peripheral cells(i.e., PBMCs or lymph node cells). Such regulatory activities of TY21580on T cells provide a mechanistic understanding for the potent in vivoanti-tumor efficacy of TY21580. Without wishing to be bound by theory,these results suggest that TY21580 reduces immunosuppressive Tregactivity and enhances cytotoxic T lymphocyte (CD8⁺ T cell) activity inthe tumor microenvironment to mediate anti-tumor responses.

Example 19: TY21580 Anti-Tumor Efficacy in a Large Established H22 LiverTumor Model

To determine the anti-tumor efficacy of TY21580 in large establishedtumors, female BALB/c mice were inoculated subcutaneously with mouse H22liver cancer cells. When relatively large tumors were established ateither ˜500 mm³ or ˜800 mm³, mice were treated with TY21580 at 5 mg/kgby intraperitoneal injection twice a week (BIW) for 4 doses. An isotypeantibody was used as a control. Group averaged tumor growth (FIG. 64A)and individual tumor growth of each mouse in different groups (FIGS.64B-64D) was monitored twice a week and reported as the mean tumorvolume±SEM over time.

As shown in FIG. 64A for group averaged tumor growth and FIGS. 64B-64Dfor individual tumor growth, significant regression of the establishedlarge H22 tumors was observed in both TY21580 treatment groups (i.e.,groups where treatment began when tumors reached a size of either ˜500mm³ as depicted in FIGS. 64A and 64C or ˜800 mm³ as depicted in FIGS.64A and 64D) as compared to the isotype control antibody treatment group(FIGS. 64A and 64B). These results demonstrate the striking efficacy ofTY21580 in the suppression of large established tumors.

Example 20: Effect of the Length of Masking Peptides on MaskingEfficiency

Two activatable antibodies, TY22402 and TY22404, were chosen to test thedependence of masking efficiency on the length of masking peptides tosuit their specific applications. The masking peptides of TY22402 andTY22404 were shortened from 21 residues to 16 or 12 residues by removingthe residues from the N-terminus, leaving only 5 or 2 residues beforethe first cysteine residue in the masking peptide (Table 26). Theseactivatable antibodies were expressed and purified from mammalian cellsand their masking efficiencies were measured as described in Example 8and compared to parent antibody TY21580. Results from two experimentsindicated that these activatable antibodies can be made using differentmasking peptides with lengths ranging from 2 to 11 residues before thefirst cysteine residue to modulate antibody masking efficiency (FIGS.65A and 65B; Tables 27 and 28). This seems to suggest that the coremasking motif contains the cysteine loop and its immediately adjacentresidues, and is sufficient to maintain masking efficiency.

TABLE 26 Masking peptides with varying peptide lengths Sample ID:Masking + cleavage peptide sequences (underlined): TY22402EVGSYIVHHSDCDAFYPYCDSSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 197) TY22775EVGHSDCDAFYPYCDSSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 198) TY22864EDCDAFYPYCDSSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 199) TY22404EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 200) TY22776EVGSSDCVPYYYACAYSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 201) TY22871EDCVPYYYACAYSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 202)

Table 27 shows the masking efficiencies of the antibodies in FIG. 65A.Table 28 shows the masking efficiencies of the antibodies in FIG. 65B.

TABLE 27 Masking efficiencies of antibodies with varying masking peptidelengths Masking Sample ID EC50(nM) efficiency TY21580 0.2223 TY2240253.99 243 TY22775 37.31 168 TY22404 68.40 308 TY22776 65.90 296

TABLE 28 Masking efficiencies of antibodies with varying masking peptidelengths Masking Sample ID EC50(nM) efficiency TY21580 0.2125 TY22402115.6 554 TY22864 117 550 TY22404 121.5 572 TY22871 88.09 414

Example 21: Effect of the Length of Cleavage Peptides on MaskingEfficiency

TY22404 was chosen to test the dependence of masking efficiency on thelength of the cleavage peptide to suit their specific applications. Thecleavage peptide of TY22404 was shortened to various lengths (Table 29).Activatable antibodies were expressed and purified from mammalian cells,and their masking efficiencies were measured as described in Example 8and compared to parent antibody TY21580. As shown in FIG. 66 and Table30, the results indicated that these activatable antibodies can be madeusing different cleavage peptides with their length ranging from 5 to 20residues to modulate antibody masking efficiency. The strong correlationbetween masking and cleavage motifs is striking; the masking efficiencyof TY23291 is enhanced at least 30-fold compared to TY22404 when thepeptide length is truncated from 41 to 17 amino acids. These resultsindicate that several novel masking peptides can be designed andengineered. In addition, the coupling between masking and cleavagemotifs could be further explored.

TABLE 29 Masking peptides with varying cleavage peptide lengths SamplePeptide Masking + ID name cleavage peptide sequences (underlined):TY22404 EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTPLGLAGS GGS (SEQ ID NO: 200)TY23286 EVGSYPNPSSDCVPYYYACAYSGRSAPLGLA (SEQ ID NO: 209) TY23289EDCVPYYYACAYSGRSAPLGLA (SEQ ID NO: 210) TY23280 EDCVPYYYACAYSGRSA(SEQ ID NO: 211) TY23291 EDCVPYYYACAYPLGLA (SEQ ID NO: 212)

Table 30 shows the masking efficiencies of the antibodies in FIG. 66.

TABLE 30 Masking efficiencies of antibodies with varying cleavagepeptide lengths Masking Sample ID EC50 (nM) efficiency TY21580 0.2505TY22404 117.4 469 TY23286 1496 5972 TY23289 133.2 532 TY23280 2952 11784TY23291 3656 14595

What is claimed is:
 1. An activatable antibody comprising: a firstpolypeptide comprising, from N-terminus to C-terminus, a masking moiety(MM), a cleavable moiety (CM), and a target binding moiety (TBM),wherein the MM comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 189-193, 195-196, and 213-216; wherein the CMcomprises at least a first cleavage site; wherein: a) the TBM comprisesan antibody light chain variable region (VL), and the activatableantibody further comprises a second polypeptide comprising an antibodyheavy chain variable region (VH); b) the TBM comprises an antibody heavychain variable region (VH), and the activatable antibody furthercomprises a second polypeptide comprising an antibody light chainvariable region (VL); c) the TBM comprises from the N-terminus to theC-terminus, an antibody light chain variable region (VL) and an antibodyheavy chain variable region (VH); or d) the TBM comprises from theN-terminus to the C-terminus, an antibody heavy chain variable region(VH) and an antibody light chain variable region (VL); wherein the VHcomprises an HVR-H1 comprising the amino acid sequence YSISSGYHWSWI (SEQID NO: 23), an HVR-H2 comprising the amino acid sequenceLARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35), and an HVR-H3 comprising the aminoacid sequence ARSYVYFDY (SEQ ID NO: 45); wherein the VL comprises anHVR-L1 comprising the amino acid sequence RASQSVRGRFLA (SEQ ID NO: 58),an HVR-L2 comprising the amino acid sequence DASNRATGI (SEQ ID NO: 66),and an HVR-L3 comprising the amino acid sequence YCQQSSSWPPT (SEQ ID NO:75); and wherein the activatable antibody binds to human CTLA4 via theVH and VL when the CM is cleaved.
 2. The activatable antibody of claim1, wherein the TBM comprises an antibody light chain variable region(VL), and the activatable antibody further comprises a secondpolypeptide comprising an antibody heavy chain variable region (VH). 3.The activatable antibody of claim 1, wherein the first cleavage site isa protease cleavage site for a protease selected from the groupconsisting of urokinase-type plasminogen activator (uPA), matrixmetalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, TobaccoEtch Virus (TEV) protease, plasmin, Thrombin, Factor X, PSA, PSMA,Cathepsin D, Cathepsin K, Cathepsin S, ADAM10, ADAM12, ADAMTS,Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6,Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12,Caspase-13, Caspase-14, and TACE.
 4. The activatable antibody of claim1, wherein the CM further comprises a first linker (L₁) C-terminal tothe first cleavage site.
 5. The activatable antibody of claim 4, whereinthe L₁ comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 156-163.
 6. The activatable antibody of claim1, wherein the CM further comprises a second cleavage site.
 7. Theactivatable antibody of claim 6, wherein the CM further comprises afirst linker (L₁) C-terminal to the first cleavage site, wherein thesecond cleavage site is C-terminal to the L₁.
 8. The activatableantibody of claim 6, wherein the second cleavage site is a proteasecleavage site for a protease selected from the group consisting ofurokinase-type plasminogen activator (uPA), matrix metalloproteinase-1(MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, Tobacco Etch Virus (TEV)protease, plasmin, Thrombin, Factor X, PSA, PSMA, Cathepsin D, CathepsinK, Cathepsin S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3,Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9,Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE. 9.The activatable antibody of claim 6, wherein the first and secondcleavage sites are different.
 10. The activatable antibody of claim 6,wherein the CM further comprises a second linker (L₂) C-terminal to thesecond cleavage site.
 11. The activatable antibody of claim 10, whereinthe L₂ comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 156-163.
 12. The activatable antibody of claim1, wherein the CM further comprises a linker (L₃) N-terminal to thefirst cleavage site.
 13. The activatable antibody of claim 1, whereinthe CM comprises at least a first protease cleavage site and is cleavedwith one or more proteases selected from the group consisting ofurokinase-type plasminogen activator (uPA), matrix metalloproteinase-1(MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, Tobacco Etch Virus (TEV)protease, plasmin, Thrombin, Factor X, PSA, PSMA, Cathepsin D, CathepsinK, Cathepsin S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3,Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9,Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE.14. The activatable antibody of claim 1, wherein the activatableantibody comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 165-179 and 198-202.
 15. A method of treatingor delaying progression of cancer in a subject in need thereof, themethod comprising administering to the subject an effective amount ofthe activatable antibody of claim
 1. 16. A method of reducing size of asolid tumor in a subject in need thereof, wherein the solid tumor has asize of about 400-1000 mm³, and the method comprises administering tothe subject an effective amount of the activatable antibody of claim 1.17. The method of claim 16, further comprising administering to thesubject an effective amount of at least one additional therapeuticagent, wherein the at least one additional therapeutic agent is selectedfrom the group consisting of viral gene therapy, immune checkpointinhibitors, target therapies, radiation therapies, vaccinationtherapies, and chemotherapies.
 18. The activatable antibody of claim 1,wherein the VH comprises the amino acid sequence of SEQ ID NO: 87, andthe VL comprises the amino acid sequence of SEQ ID NO:
 100. 19. Theactivatable antibody of claim 1, wherein the MM comprises the amino acidsequence PNPSSDCVPYYYACAY (SEQ ID NO: 144).
 20. The activatable antibodyof claim 18, wherein the MM comprises the amino acid sequencePNPSSDCVPYYYACAY (SEQ ID NO: 144).
 21. The activatable antibody of claim1, wherein the activatable antibody comprises the amino acid sequenceEVGSYPNPSSDCVPYYYACAY (SEQ ID NO: 192).
 22. The activatable antibody ofclaim 18, wherein the activatable antibody comprises the amino acidsequence EVGSYPNPSSDCVPYYYACAY (SEQ ID NO: 192).
 23. An activatableantibody comprising: (a) a first polypeptide comprising, from N-terminusto C-terminus, a masking moiety (MM), a cleavable moiety (CM), and anantibody light chain variable region (VL); and (b) a second polypeptidecomprising the antibody heavy chain variable region (VH); wherein the MMcomprises the amino acid sequence PNPSSDCVPYYYACAY (SEQ ID NO: 144);wherein the CM comprises at least a first cleavage site; wherein the VHcomprises the amino acid sequence of SEQ ID NO: 87, and wherein the VLcomprises the amino acid sequence of SEQ ID NO: 100; and wherein theactivatable antibody binds to human CTLA4 via the VH and VL when the CMis cleaved.
 24. The activatable antibody of claim 23, wherein the firstpolypeptide comprises the amino acid sequenceEVGSYPNPSSDCVPYYYACAYSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 200).
 25. Theactivatable antibody of claim 1, wherein the activatable antibodycomprises a human IgG1 Fc region comprising one or more mutations thatincrease antibody-dependent cellular cytotoxicity (ADCC) activity. 26.The activatable antibody of claim 23, wherein the activatable antibodycomprises a human IgG1 Fc region comprising one or more mutations thatincrease antibody-dependent cellular cytotoxicity (ADCC) activity.
 27. Amethod of treating or delaying progression of cancer in a subject inneed thereof, the method comprising administering to the subject aneffective amount of the activatable antibody of claim
 18. 28. A methodof treating or delaying progression of cancer in a subject in needthereof, the method comprising administering to the subject an effectiveamount of the activatable antibody of claim
 19. 29. A method of treatingor delaying progression of cancer in a subject in need thereof, themethod comprising administering to the subject an effective amount ofthe activatable antibody of claim
 20. 30. A method of treating ordelaying progression of cancer in a subject in need thereof, the methodcomprising administering to the subject an effective amount of theactivatable antibody of claim
 21. 31. A method of treating or delayingprogression of cancer in a subject in need thereof, the methodcomprising administering to the subject an effective amount of theactivatable antibody of claim
 23. 32. A method of treating or delayingprogression of cancer in a subject in need thereof, the methodcomprising administering to the subject an effective amount of theactivatable antibody of claim
 24. 33. A method of treating or delayingprogression of cancer in a subject in need thereof, the methodcomprising administering to the subject an effective amount of theactivatable antibody of claim
 25. 34. A pharmaceutical compositioncomprising the activatable antibody of claim 1 and a pharmaceuticallyacceptable carrier.
 35. A pharmaceutical composition comprising theactivatable antibody of claim 18 and a pharmaceutically acceptablecarrier.
 36. A pharmaceutical composition comprising the activatableantibody of claim 21 and a pharmaceutically acceptable carrier.
 37. Apharmaceutical composition comprising the activatable antibody of claim22 and a pharmaceutically acceptable carrier.
 38. A pharmaceuticalcomposition comprising the activatable antibody of claim 23 and apharmaceutically acceptable carrier.
 39. A pharmaceutical compositioncomprising the activatable antibody of claim 24 and a pharmaceuticallyacceptable carrier.
 40. A pharmaceutical composition comprising theactivatable antibody of claim 25 and a pharmaceutically acceptablecarrier.